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1
Denome, R. M., and C. N. Cole. "Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals." Molecular and Cellular Biology 8, no. 11 (November 1988): 4829–39. http://dx.doi.org/10.1128/mcb.8.11.4829.
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We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.
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Denome, R. M., and C. N. Cole. "Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals." Molecular and Cellular Biology 8, no. 11 (November 1988): 4829–39. http://dx.doi.org/10.1128/mcb.8.11.4829-4839.1988.
Full textAbstract:
We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.
3
Stolow, D. T., and S. M. Berget. "UV cross-linking of polypeptides associated with 3'-terminal exons." Molecular and Cellular Biology 10, no. 11 (November 1990): 5937–44. http://dx.doi.org/10.1128/mcb.10.11.5937.
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Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.
4
Stolow, D. T., and S. M. Berget. "UV cross-linking of polypeptides associated with 3'-terminal exons." Molecular and Cellular Biology 10, no. 11 (November 1990): 5937–44. http://dx.doi.org/10.1128/mcb.10.11.5937-5944.1990.
Full textAbstract:
Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.
5
Sanfaçon, Hélène. "Regulation of mRNA formation in plants: lessons from the cauliflower mosaic virus transcription signals." Canadian Journal of Botany 70, no. 5 (May 1, 1992): 885–99. http://dx.doi.org/10.1139/b92-113.
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The cauliflower mosaic virus (CaMV) transcription signals are common tools of plant molecular biologists. In this article, the transcription signals are discussed in light of the life cycle of CaMV, a plant pararetrovirus. Production of mature 35S RNA, the terminally redundant genomic RNA, is regulated by the 35S promoter, a very strong promoter, and by the polyadenylation signal that is present twice on the RNA but recognized only at its 3′ end. Dissection of the promoter has identified several organ-specific elements acting in concert to express the 35S RNA in most plant cells. Studies on the polyadenylation signal have revealed upstream elements inducing recognition of the AATAAA sequence and have led to the proposal that the conditional recognition of this signal is dependent on its distance from the promoter. Comparison of the CaMV signals with other plant signals allows speculation on the plant transcriptional machinery and on some striking resemblances and differences to the animal and yeast systems. Finally, potential applications of this knowledge will be described such as the construction of hybrid plant promoters or polyadenylation signals using the 35S minimal elements. Key words: cauliflower mosaic virus, 35S promoter, polyadenylation signal, 35S RNA, transcription, retroviruses.
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Das, Atze T., Bep Klaver, and Ben Berkhout. "A Hairpin Structure in the R Region of the Human Immunodeficiency Virus Type 1 RNA Genome Is Instrumental in Polyadenylation Site Selection." Journal of Virology 73, no. 1 (January 1, 1999): 81–91. http://dx.doi.org/10.1128/jvi.73.1.81-91.1999.
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ABSTRACT Some retroviruses with an extended repeat (R) region encode the polyadenylation signal within the R region such that this signal is present at both the 5′ and 3′ ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5′ polyadenylation signal or enhance usage of the 3′ signal. The human immunodeficiency virus type 1 (HIV-1) genome encodes an inherently efficient polyadenylation signal within the 97-nucleotide R region. Polyadenylation at the 5′ HIV-1 polyadenylation site is inhibited by downstream splicing signals, and usage of the 3′ polyadenylation site is triggered by an upstream enhancer element. In this paper, we demonstrate that this on-off switch of the HIV-1 polyadenylation signal is controlled by a secondary RNA structure that occludes part of the AAUAAA hexamer motif, which we have termed the polyA hairpin. Opening the 5′ hairpin by mutation triggered premature polyadenylation and caused reduced synthesis of viral RNA, indicating that the RNA structure plays a pivotal role in repression of the 5′ polyadenylation site. Apparently, the same hairpin structure does not interfere with efficient usage of the 3′ polyadenylation site, which may be due to the presence of the upstream enhancer element. However, when the 3′ hairpin was further stabilized by mutation, we measured a complete loss of 3′ polyadenylation. Thus, the thermodynamic stability of the polyA hairpin is delicately balanced to allow nearly complete repression of the 5′ site yet efficient activation of the 3′ site. This is the first report of regulated polyadenylation that is mediated by RNA secondary structure. A similar hairpin motif that occludes the polyadenylation signal can be proposed for other lentiviruses and members of the spumaretroviruses, suggesting that this represents a more general gene expression strategy of complex retroviruses.
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Rothnie, Helen M., Gang Chen, Johannes Fütterer, and Thomas Hohn. "Polyadenylation in Rice Tungro Bacilliform Virus:cis-Acting Signals and Regulation." Journal of Virology 75, no. 9 (May 1, 2001): 4184–94. http://dx.doi.org/10.1128/jvi.75.9.4184-4194.2001.
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ABSTRACT The polyadenylation signal of rice tungro bacilliform virus (RTBV) was characterized by mutational and deletion analysis. Thecis-acting signals required to direct polyadenylation conformed to what is known for plant poly(A) signals in general and were very similar to those of the related cauliflower mosaic virus. Processing was directed by a canonical AAUAAA poly(A) signal, an upstream UG-rich region considerably enhanced processing efficiency, and sequences downstream of the cleavage site were not required. When present at the end of a transcription unit, thecis-acting signals for 3′-end processing were highly efficient in both monocot (rice) and dicot (Nicotiana plumbaginifolia) protoplasts. In a promoter-proximal position, as in the viral genome, the signal was also efficiently processed in rice protoplasts, giving rise to an abundant “short-stop” (SS-) RNA. The proportion of SS-RNA was considerably lower in N. plumbaginifolia protoplasts. In infected plants, SS-RNA was hardly detectable, suggesting either that SS-RNA is unstable in infected plants or that read-through of the promoter-proximal poly(A) site is very efficient. SS-RNA is readily detectable in transgenic rice plants (A. Klöti, C. Henrich, S. Bieri, X. He, G. Chen, P. K. Burkhardt, J. Wünn, P. Lucca, T. Hohn, I. Potrylus, and J. Fütterer, 1999. Plant Mol. Biol. 40:249–266), thus the absence of SS-RNA in infected plants can be attributed to poly(A) site bypass in the viral context to ensure production of the full-length pregenomic viral RNA. RTBV poly(A) site suppression thus depends both on context and the expression system; our results suggest that the circular viral minichromosome directs assembly of a transcription-processing complex with specific properties to effect read-through of the promoter-proximal poly(A) signal.
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Butler, J. S., P. P. Sadhale, and T. Platt. "RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs." Molecular and Cellular Biology 10, no. 6 (June 1990): 2599–605. http://dx.doi.org/10.1128/mcb.10.6.2599.
Full textAbstract:
Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.
9
Butler, J. S., P. P. Sadhale, and T. Platt. "RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs." Molecular and Cellular Biology 10, no. 6 (June 1990): 2599–605. http://dx.doi.org/10.1128/mcb.10.6.2599-2605.1990.
Full textAbstract:
Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.
10
Castelo-Branco, Pedro, Andre Furger, Matthew Wollerton, Christopher Smith, Alexandra Moreira, and Nick Proudfoot. "Polypyrimidine Tract Binding Protein Modulates Efficiency of Polyadenylation." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4174–83. http://dx.doi.org/10.1128/mcb.24.10.4174-4183.2004.
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ABSTRACT Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3′ end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3′ end processing.
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Takagaki, Y., and J. L. Manley. "RNA recognition by the human polyadenylation factor CstF." Molecular and Cellular Biology 17, no. 7 (July 1997): 3907–14. http://dx.doi.org/10.1128/mcb.17.7.3907.
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Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.
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Cooke, Charles, and James C. Alwine. "Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal." Molecular and Cellular Biology 22, no. 13 (July 1, 2002): 4579–86. http://dx.doi.org/10.1128/mcb.22.13.4579-4586.2002.
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ABSTRACT Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC′, AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC′ complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC′ is a precursor of AC, and the AC′ and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC′ complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC′ complex is dependent on the polypyrimidine tract. The transition from AC′ to AC appears to require an intact 3′-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC′ complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.
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Hyman, L. E., and C. L. Moore. "Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites." Molecular and Cellular Biology 13, no. 9 (September 1993): 5159–67. http://dx.doi.org/10.1128/mcb.13.9.5159.
Full textAbstract:
Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.
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Hyman, L. E., and C. L. Moore. "Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites." Molecular and Cellular Biology 13, no. 9 (September 1993): 5159–67. http://dx.doi.org/10.1128/mcb.13.9.5159-5167.1993.
Full textAbstract:
Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.
15
Zhao, Xiaomin, Daniel Öberg, Margaret Rush, Joanna Fay, Helen Lambkin, and Stefan Schwartz. "A 57-Nucleotide Upstream Early Polyadenylation Element in Human Papillomavirus Type 16 Interacts with hFip1, CstF-64, hnRNP C1/C2, and Polypyrimidine Tract Binding Protein." Journal of Virology 79, no. 7 (April 1, 2005): 4270–88. http://dx.doi.org/10.1128/jvi.79.7.4270-4288.2005.
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ABSTRACT We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3′ UTR) in HPV-16 gene expression. We found that deletion of the early 3′ UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3′ half of the early 3′ UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3′ splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.
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Jin, Jianping, and Linda A. Guarino. "3′-End Formation of Baculovirus Late RNAs." Journal of Virology 74, no. 19 (October 1, 2000): 8930–37. http://dx.doi.org/10.1128/jvi.74.19.8930-8937.2000.
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ABSTRACT Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mechanism of 3′-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3′-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3′ ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism.
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Raju, Ramaswamy, Mustapha Hajjou, Kristie R. Hill, Vandana Botta, and Sisir Botta. "In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway." Journal of Virology 73, no. 3 (March 1, 1999): 2410–19. http://dx.doi.org/10.1128/jvi.73.3.2410-2419.1999.
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ABSTRACT Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.
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Sisodia, S. S., B. Sollner-Webb, and D. W. Cleveland. "Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation." Molecular and Cellular Biology 7, no. 10 (October 1987): 3602–12. http://dx.doi.org/10.1128/mcb.7.10.3602.
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To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
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Sisodia, S. S., B. Sollner-Webb, and D. W. Cleveland. "Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation." Molecular and Cellular Biology 7, no. 10 (October 1987): 3602–12. http://dx.doi.org/10.1128/mcb.7.10.3602-3612.1987.
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To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
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Sena, Rebecca M., Jeffery L. Twiss, Amy S. Gardiner, Michela Dell’Orco, David N. Linsenbardt, and Nora I. Perrone-Bizzozero. "The RNA-Binding Protein HuD Regulates Alternative Splicing and Alternative Polyadenylation in the Mouse Neocortex." Molecules 26, no. 10 (May 11, 2021): 2836. http://dx.doi.org/10.3390/molecules26102836.
Full textAbstract:
The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3′-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4–/– (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3′-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.
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Qian, Z. W., and J. Wilusz. "An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal." Molecular and Cellular Biology 11, no. 10 (October 1991): 5312–20. http://dx.doi.org/10.1128/mcb.11.10.5312.
Full textAbstract:
We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.
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Qian, Z. W., and J. Wilusz. "An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal." Molecular and Cellular Biology 11, no. 10 (October 1991): 5312–20. http://dx.doi.org/10.1128/mcb.11.10.5312-5320.1991.
Full textAbstract:
We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.
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Stebbins-Boaz, B., and J. D. Richter. "Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development." Molecular and Cellular Biology 14, no. 9 (September 1994): 5870–80. http://dx.doi.org/10.1128/mcb.14.9.5870.
Full textAbstract:
Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.
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Stebbins-Boaz, B., and J. D. Richter. "Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development." Molecular and Cellular Biology 14, no. 9 (September 1994): 5870–80. http://dx.doi.org/10.1128/mcb.14.9.5870-5880.1994.
Full textAbstract:
Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly." Molecular and Cellular Biology 9, no. 4 (April 1989): 1759–71. http://dx.doi.org/10.1128/mcb.9.4.1759.
Full textAbstract:
To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly." Molecular and Cellular Biology 9, no. 4 (April 1989): 1759–71. http://dx.doi.org/10.1128/mcb.9.4.1759-1771.1989.
Full textAbstract:
To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.
27
Zhang, F., and C. N. Cole. "Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA." Molecular and Cellular Biology 7, no. 9 (September 1987): 3277–86. http://dx.doi.org/10.1128/mcb.7.9.3277.
Full textAbstract:
Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.
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Zhang, F., and C. N. Cole. "Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA." Molecular and Cellular Biology 7, no. 9 (September 1987): 3277–86. http://dx.doi.org/10.1128/mcb.7.9.3277-3286.1987.
Full textAbstract:
Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.
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Beaudet, Lucille, Guy Charron, and Jean-Pierre Julien. "Origin of the two mRNA species for the human neurofilament light gene." Biochemistry and Cell Biology 70, no. 5 (May 1, 1992): 279–84. http://dx.doi.org/10.1139/o92-044.
Full textAbstract:
The human neurofilament light (hNF-L) gene yields two major species of mRNAs of 2.4 and 3.8 kilobases (kb) in size. To investigate the origin of these two mRNAs, we have analyzed in transgenic mice the expression of hNF-L DNA fragments including different lengths of 5′ -flanking regions. The finding that the 3.8-kb mRNA species is produced by a hNF-L transgene that includes only the proximal promoter region (−0.3 kb) demonstrates that both the 2.4- and 3.8-kb mRNAs are derived from the same site of transcription initiation. Sequencing of the 3′ untranslated region of the hNF-L gene revealed the presence of multiple AATAAA polyadenylation signals. We conclude from Northern blotting experiments using probes spanning various regions of the hNF-L gene that the 2.4- and 3.8-kb mRNAs originate from the selective use of polyadenylation signals located 1.4 kb apart.Key words: neurofilament gene, polyadenylation, transgenic mice, intermediate filament gene, RNA processing.
30
Sadhale, P. P., and T. Platt. "Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 10 (October 1992): 4262–70. http://dx.doi.org/10.1128/mcb.12.10.4262.
Full textAbstract:
A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements. Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA. Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition. Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed. We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme. By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing. This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose. As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro. Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.
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Sadhale, P. P., and T. Platt. "Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 10 (October 1992): 4262–70. http://dx.doi.org/10.1128/mcb.12.10.4262-4270.1992.
Full textAbstract:
A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements. Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA. Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition. Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed. We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme. By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing. This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose. As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro. Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.
32
Zaiss, Anne-Kathrin, Sodany Son, and Lung-Ji Chang. "RNA 3′ Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy." Journal of Virology 76, no. 14 (July 15, 2002): 7209–19. http://dx.doi.org/10.1128/jvi.76.14.7209-7219.2002.
Full textAbstract:
ABSTRACT The expression of reporter genes driven by the same human elongation factor 1α (EF1α) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1α intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1α transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1α intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3′-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3′ polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.
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Cooke, Charles, Holly Hans, and James C. Alwine. "Utilization of Splicing Elements and Polyadenylation Signal Elements in the Coupling of Polyadenylation and Last-Intron Removal." Molecular and Cellular Biology 19, no. 7 (July 1, 1999): 4971–79. http://dx.doi.org/10.1128/mcb.19.7.4971.
Full textAbstract:
ABSTRACT Polyadenylation (PA) is the process by which the 3′ ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3′ splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5′ splice site in the presence of a viable polypyrimidine tract and the 3′ splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5′ splice site) within the last exon can negatively effect both PA and splicing; hence, a 5′ splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.
34
Luo, Y., and G. G. Carmichael. "Splice site choice in a complex transcription unit containing multiple inefficient polyadenylation signals." Molecular and Cellular Biology 11, no. 10 (October 1991): 5291–300. http://dx.doi.org/10.1128/mcb.11.10.5291.
Full textAbstract:
The relationship between polyadenylation and splicing was investigated in a model system consisting of two tandem but nonidentical polyomavirus late transcription units. This model system exploits the polyomavirus late transcription termination and polyadenylation signals, which are sufficiently weak to allow the production of many multigenome-length primary transcripts with repeating introns, exons, and poly(A) sites. This double-genome construct contains exons of two types, those bordered by 3' and 5' splice sites (L1 and L2) and those bordered by a 3' splice site and a poly(A) site (V1 and V2). The L1 and L2 exons are distinguishable from one another but retain identical flanking RNA processing signals, as is the case for the V1 and V2 exons. Analysis of cytoplasmic RNAs obtained from mouse cells transfected with this construct and its derivatives revealed the following. (i) V1 and V2 exons are often skipped during pre-mRNA processing, while L1 and L2 exons are not skipped. (ii) No messages contain internal, unused polyadenylation signals. (iii) Poly(A) site choice is not required for the selection of an upstream 3' splice site. (iv) When two tandem poly(A) sites are placed downstream of a 3' splice site, the first poly(A) site is chosen almost exclusively, even though transcription can proceed past both sites. (v) Placing a 3' splice site between these two tandem poly(A) sites allows the more distal site to be chosen. These and other available data are most consistent with a model in which terminal exons are produced by the coordinate selection and use of a 3' splice site with the nearest available downstream poly(A) site.
35
Luo, Y., and G. G. Carmichael. "Splice site choice in a complex transcription unit containing multiple inefficient polyadenylation signals." Molecular and Cellular Biology 11, no. 10 (October 1991): 5291–300. http://dx.doi.org/10.1128/mcb.11.10.5291-5300.1991.
Full textAbstract:
The relationship between polyadenylation and splicing was investigated in a model system consisting of two tandem but nonidentical polyomavirus late transcription units. This model system exploits the polyomavirus late transcription termination and polyadenylation signals, which are sufficiently weak to allow the production of many multigenome-length primary transcripts with repeating introns, exons, and poly(A) sites. This double-genome construct contains exons of two types, those bordered by 3' and 5' splice sites (L1 and L2) and those bordered by a 3' splice site and a poly(A) site (V1 and V2). The L1 and L2 exons are distinguishable from one another but retain identical flanking RNA processing signals, as is the case for the V1 and V2 exons. Analysis of cytoplasmic RNAs obtained from mouse cells transfected with this construct and its derivatives revealed the following. (i) V1 and V2 exons are often skipped during pre-mRNA processing, while L1 and L2 exons are not skipped. (ii) No messages contain internal, unused polyadenylation signals. (iii) Poly(A) site choice is not required for the selection of an upstream 3' splice site. (iv) When two tandem poly(A) sites are placed downstream of a 3' splice site, the first poly(A) site is chosen almost exclusively, even though transcription can proceed past both sites. (v) Placing a 3' splice site between these two tandem poly(A) sites allows the more distal site to be chosen. These and other available data are most consistent with a model in which terminal exons are produced by the coordinate selection and use of a 3' splice site with the nearest available downstream poly(A) site.
36
Qiu, Jianming, Ramnath Nayak, and David J. Pintel. "Alternative Polyadenylation of Adeno-Associated Virus Type 5 RNA within an Internal Intron Is Governed by both a Downstream Element within the Intron 3′ Splice Acceptor and an Element Upstream of the P41 Initiation Site." Journal of Virology 78, no. 1 (January 1, 2004): 83–93. http://dx.doi.org/10.1128/jvi.78.1.83-93.2004.
Full textAbstract:
ABSTRACT Adeno-associated virus type 5 (AAV5) has a linear, single-stranded DNA genome of ca. 5 kb and an overlapping transcription profile featuring multiple promoters and a single intron in the center of the genome. Unlike the situation for the prototype AAV2, AAV5 RNAs transcribed from upstream promoters at map units 7 (P7) and 19 (P19), which encode the viral Rep proteins, are predominantly polyadenylated at a site within the intron [(pA)p]. RNAs generated from the AAV5 capsid gene promoter P41, which is only 78 nucleotides (nt) upstream of the intron donor, and 281 nt upstream of (pA)p, primarily readthrough (pA)p, are polyadenylated at a more distal site at the 3′ end of the genome [(pA)d] and ultimately spliced. The intron contains the core sequences sufficient for polyadenylation at (pA)p, which is governed by a G/U-rich downstream element that overlaps with the intron 3′ A2 splice acceptor. In addition, polyadenylation of P7- and P19-generated RNAs at (pA)p is influenced by an upstream element that lies 5′ to the start of the P41 transcript. Our results also suggest that splicing and polyadenylation of P41-generated RNA can compete for the same pool of precursor pre-mRNA molecules. The cis-acting signals within the A2 3′ splice site that govern polyadenylation and splicing of AAV5 RNAs must be optimized to program both (i) the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to generate the proper levels of the essential Rep proteins and (ii) the splicing of P41-generated RNAs to generate the proper ratio of capsid proteins during AAV5 infection.
37
Sun, Xiaojie, Ji Li, Xun Sun, Wanqi Liu, and Xiangwei Meng. "CFIm25 in Solid Tumors: Current Research Progress." Technology in Cancer Research & Treatment 19 (January 1, 2020): 153303382093396. http://dx.doi.org/10.1177/1533033820933969.
Full textAbstract:
Cleavage factor I m25 is a newly discovered solid tumor-related gene, however, its precise role in cancer pathogenesis has not yet been characterized. Alternative polyadenylation is an RNA-processing mechanism that generates distinct 3′-termini on messenger RNAs, producing messenger RNA isoforms. Different factors influence the initiation and development of this process. As a key factor in alternative polyadenylation, cleavage factor I m25 plays an important role in messenger RNA maturation and cell signal transduction. Moreover, by regulating the process of alternative polyadenylation, it can inhibit the proliferation, invasion, and metastasis of a variety of tumors. Cleavage factor I m25 also acts as an oncogene in select tumors. The present review focuses on the role of cleavage factor I m25 in solid tumors and treatment. Due to the lack of current knowledge regarding the mechanisms of action and regulation of cleavage factor I m25 and alternative polyadenylation, it is necessary to further examine their role in cancer as well as in other diseases.
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Steinmetz, Eric J., Sarah B. H. Ng, Joseph P. Cloute, and David A. Brow. "cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II." Molecular and Cellular Biology 26, no. 7 (April 1, 2006): 2688–96. http://dx.doi.org/10.1128/mcb.26.7.2688-2696.2006.
Full textAbstract:
ABSTRACT Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3′-end formation in the context of an mRNA 3′ untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.
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Zhao, Jing, Linda Hyman, and Claire Moore. "Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis." Microbiology and Molecular Biology Reviews 63, no. 2 (June 1, 1999): 405–45. http://dx.doi.org/10.1128/mmbr.63.2.405-445.1999.
Full textAbstract:
SUMMARY Formation of mRNA 3′ ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3′ ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.
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Terhune, Scott S., Christine Milcarek, and Laimonis A. Laimins. "Regulation of Human Papillomavirus Type 31 Polyadenylation during the Differentiation-Dependent Life Cycle." Journal of Virology 73, no. 9 (September 1, 1999): 7185–92. http://dx.doi.org/10.1128/jvi.73.9.7185-7192.1999.
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ABSTRACT The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3′ ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3′ ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.
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Nie, Xingcao, Jinhong Chang, and John M. Taylor. "Alternative Processing of Hepatitis Delta Virus Antigenomic RNA Transcripts." Journal of Virology 78, no. 9 (May 1, 2004): 4517–24. http://dx.doi.org/10.1128/jvi.78.9.4517-4524.2004.
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ABSTRACT Intrinsic to the life cycle of hepatitis delta virus (HDV) is the fact that its RNAs undergo different forms of posttranscriptional RNA processing. Transcripts of both the genomic RNA and its exact complement, the antigenomic RNA, undergo ribozyme cleavage and RNA ligation. In addition, antigenomic RNA transcripts can undergo 5′ capping, 3′ polyadenylation, and even RNA editing by an adenosine deaminase. This study focused on the processing of antigenomic RNA transcripts. Two approaches were used to study the relationship between the events of polyadenylation, ribozyme cleavage, and RNA ligation. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing. We found that when a separate stable source of δAg-S, the small delta protein, was provided, the replication ability of the mutated RNA was restored. The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. The DNA constructs used were such that the RNA transcripts were antigenomic and began at the same 5′ site as the mRNA produced during RNA-directed HDV genome replication. A series of such constructs was assembled in order to test the relative abilities of the transcripts to undergo processing by polyadenylation or ribozyme cleavage at sites further 3′ on a multimer of HDV sequences. The findings from the two experimental approaches led to significant modifications in the rolling-circle model of HDV genome replication.
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Peros, Iván Gabriel, Carolina Susana Cerrudo, Marcela Gabriela Pilloff, Mariano Nicolás Belaich, Mario Enrique Lozano, and Pablo Daniel Ghiringhelli. "Advances in the Bioinformatics Knowledge of mRNA Polyadenylation in Baculovirus Genes." Viruses 12, no. 12 (December 6, 2020): 1395. http://dx.doi.org/10.3390/v12121395.
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Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3′ end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3′ ends from 180 baculovirus genomes.
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Sato, K., R. Ito, K. H. Baek, and K. Agarwal. "A specific DNA sequence controls termination of transcription in the gastrin gene." Molecular and Cellular Biology 6, no. 4 (April 1986): 1032–43. http://dx.doi.org/10.1128/mcb.6.4.1032.
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We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.
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Sato, K., R. Ito, K. H. Baek, and K. Agarwal. "A specific DNA sequence controls termination of transcription in the gastrin gene." Molecular and Cellular Biology 6, no. 4 (April 1986): 1032–43. http://dx.doi.org/10.1128/mcb.6.4.1032-1043.1986.
Full textAbstract:
We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.
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Majerciak, Vladimir, Koji Yamanegi, and Zhi-Ming Zheng. "Gene Structure and Expression of Kaposi's Sarcoma-Associated Herpesvirus ORF56, ORF57, ORF58, and ORF59." Journal of Virology 80, no. 24 (October 4, 2006): 11968–81. http://dx.doi.org/10.1128/jvi.01394-06.
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ABSTRACT Though similar to those of herpesvirus saimiri and Epstein-Barr virus (EBV), the Kaposi's sarcoma-associated herpesvirus (KSHV) genome features more splice genes and encodes many genes with bicistronic or polycistronic transcripts. In the present study, the gene structure and expression of KSHV ORF56 (primase), ORF57 (MTA), ORF58 (EBV BMRF2 homologue), and ORF59 (DNA polymerase processivity factor) were analyzed in butyrate-activated KSHV+ JSC-1 cells. ORF56 was expressed at low abundance as a bicistronic ORF56/57 transcript that utilized the same intron, with two alternative branch points, as ORF57 for its RNA splicing. ORF56 was transcribed from two transcription start sites, nucleotides (nt) 78994 (minor) and 79075 (major), but selected the same poly(A) signal as ORF57 for RNA polyadenylation. The majority of ORF56 and ORF57 transcripts were cleaved at nt 83628, although other nearby cleavage sites were selectable. On the opposite strand of the viral genome, colinear ORF58 and ORF59 were transcribed from different transcription start sites, nt 95821 (major) or 95824 (minor) for ORF58 and nt 96790 (minor) or 96794 (major) for ORF59, but shared overlapping poly(A) signals at nt 94492 and 94488. Two cleavage sites, at nt 94477 and nt 94469, could be equally selected for ORF59 polyadenylation, but only the cleavage site at nt 94469 could be selected for ORF58 polyadenylation without disrupting the ORF58 stop codon immediately upstream. ORF58 was expressed in low abundance as a monocistronic transcript, with a long 5′ untranslated region (UTR) but a short 3′ UTR, whereas ORF59 was expressed in high abundance as a bicistronic transcript, with a short 5′ UTR and a long 3′ UTR similar to those of polycistronic ORF60 and ORF62. Both ORF56 and ORF59 are targets of ORF57 and were up-regulated significantly in the presence of ORF57, a posttranscriptional regulator.
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Reddy, P. Seshidhar, Neeraja Idamakanti, Alexandre N. Zakhartchouk, Mohit Kumar Baxi, Joong Bok Lee, Caron Pyne, Lorne A. Babiuk, and Suresh Kumar Tikoo. "Nucleotide Sequence, Genome Organization, and Transcription Map of Bovine Adenovirus Type 3." Journal of Virology 72, no. 2 (February 1, 1998): 1394–402. http://dx.doi.org/10.1128/jvi.72.2.1394-1402.1998.
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ABSTRACT The complete DNA sequence of bovine adenovirus type 3 is reported here. The size of the genome is 34,446 bp in length with a G+C content of 54%. All the genes of the early and late regions are present in the expected locations of the genome. However, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. The deduced amino acid sequences of open reading frames (ORFs) in the late regions and early region 2 (E2) and for IVa2 show higher degrees of homology, whereas the predicted amino acid sequences of ORFs in the E1, E3, and E4 regions and the pIX, fiber, and 33,000-molecular-weight nonstructural proteins show little or no homology with the corresponding proteins of other adenoviruses. In addition, the penton base protein lacks the integrin binding motif, RGD, but has an LDV motif instead of an MDV motif. Interestingly, as in other animal adenoviruses, the virus-associated RNA genes appear to be absent from their usual location. Sequence analysis of cDNA clones representing the early- and late-region genes identified splice acceptor and splice donor sites, polyadenylation signals and polyadenylation sites, and tripartite leader sequences.
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Kan, Ming-Chung, Aparna Oruganty-Das, Amalene Cooper-Morgan, Guang Jin, Sharon A. Swanger, Gary J. Bassell, Harvey Florman, Klaus van Leyen, and Joel D. Richter. "CPEB4 Is a Cell Survival Protein Retained in the Nucleus upon Ischemia or Endoplasmic Reticulum Calcium Depletion." Molecular and Cellular Biology 30, no. 24 (October 11, 2010): 5658–71. http://dx.doi.org/10.1128/mcb.00716-10.
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ABSTRACT The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.
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Sun, Yadong, Yixiao Zhang, Keith Hamilton, James L. Manley, Yongsheng Shi, Thomas Walz, and Liang Tong. "Molecular basis for the recognition of the human AAUAAA polyadenylation signal." Proceedings of the National Academy of Sciences 115, no. 7 (December 5, 2017): E1419—E1428. http://dx.doi.org/10.1073/pnas.1718723115.
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Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3′-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3′-end formation.
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Huang, Y., and G. G. Carmichael. "Role of polyadenylation in nucleocytoplasmic transport of mRNA." Molecular and Cellular Biology 16, no. 4 (April 1996): 1534–42. http://dx.doi.org/10.1128/mcb.16.4.1534.
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To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3' end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.
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Rigo, Frank, and Harold G. Martinson. "Functional Coupling of Last-Intron Splicing and 3′-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage." Molecular and Cellular Biology 28, no. 2 (October 29, 2007): 849–62. http://dx.doi.org/10.1128/mcb.01410-07.
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ABSTRACT We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3′ splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3′-end processing of this exon can be distinguished. First, the 3′ splice site establishes connections to enhance 3′-end processing, while the nascent 3′-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3′-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3′ end, and then finishing with splicing of the last intron.