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Journal articles on the topic 'PiRNA clusters'

Author: Grafiati

Published: 25 May 2024

Last updated: 14 June 2025

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Komarov, Pavel A., Olesya Sokolova, Natalia Akulenko, Emilie Brasset, Silke Jensen, and Alla Kalmykova. "Epigenetic Requirements for Triggering Heterochromatinization and Piwi-Interacting RNA Production from Transgenes in the Drosophila Germline." Cells 9, no. 4 (2020): 922. http://dx.doi.org/10.3390/cells9040922.

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Transgenes containing a fragment of the I retrotransposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. We revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in the wild-type context, the endogenous ancestral I-related piRNAs heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. Here, we address how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. We show that a minimal amount of maternal piRNAs from ancestral I-elements is sufficient to form the transgenic piRNA clusters. Supplemental piRNAs stemming from active I-element copies do not stimulate additional chromatin changes or piRNA production from transgenes. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs, suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. It is noteworthy that the weak piRNA clusters do not transform into strong ones after being targeted by abundant I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor the formation of transgenic piRNA clusters.

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Radion, Elizaveta, Olesya Sokolova, Sergei Ryazansky, Pavel Komarov, Yuri Abramov, and Alla Kalmykova. "The Integrity of piRNA Clusters is Abolished by Insulators in the Drosophila Germline." Genes 10, no. 3 (2019): 209. http://dx.doi.org/10.3390/genes10030209.

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Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

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Chen, Peiwei, Yicheng Luo, and Alexei A. Aravin. "RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility." PLOS Genetics 17, no. 9 (2021): e1009591. http://dx.doi.org/10.1371/journal.pgen.1009591.

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piRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed “piRNA clusters” lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. RDC was thought to be absent in testis, so it remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, males lacking RDC exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis. These results disprove the common belief that RDC is dispensable for piRNA biogenesis in testis and uncover the unexpected, sexually dimorphic and dynamic behavior of a core piRNA pathway machinery.

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Assis, Raquel, and Alexey S. Kondrashov. "Rapid repetitive element-mediated expansion of piRNA clusters in mammalian evolution." Proceedings of the National Academy of Sciences 106, no. 17 (2009): 7079–82. http://dx.doi.org/10.1073/pnas.0900523106.

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Piwi-interacting RNAs (piRNAs) are ≈30 nucleotide noncoding RNAs that may be involved in transposon silencing in mammalian germline cells. Most piRNA sequences are found in a small number of genomic regions referred to as clusters, which range from 1 to hundreds of kilobases. We studied the evolution of 140 rodent piRNA clusters, 103 of which do not overlap protein-coding genes. Phylogenetic analysis revealed that 14 clusters were acquired after rat–mouse divergence and another 44 after rodent–primate divergence. Most clusters originated in a process analogous to the duplication of protein-coding genes by ectopic recombination, via insertions of long sequences that were mediated by flanking chromosome-specific repetitive elements (REs). Source sequences for such insertions are often located on the same chromosomes and also harbor clusters. The rate of piRNA cluster expansion is higher than that of any known gene family and, in contrast to other large gene families, there was not a single cluster loss. These observations suggest that piRNA cluster expansion is driven by positive selection, perhaps caused by the need to silence the ever-expanding repertoire of mammalian transposons.

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Story, Benjamin, Xing Ma, Kazue Ishihara, et al. "Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells." Life Science Alliance 2, no. 5 (2019): e201800211. http://dx.doi.org/10.26508/lsa.201800211.

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Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3′ UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

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Iyer, Shantanu S., Yidan Sun, Janine Seyfferth, et al. "The NSL complex is required for piRNA production from telomeric clusters." Life Science Alliance 6, no. 9 (2023): e202302194. http://dx.doi.org/10.26508/lsa.202302194.

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The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposonsHeT-A,TAHRE, andTART. Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. Our findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline.

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Wang, Sheng, Xiaohua Lu, Ding Qiu, and Yang Yu. "To export, or not to export: how nuclear export factor variants resolve Piwi's dilemma." Biochemical Society Transactions 49, no. 5 (2021): 2073–79. http://dx.doi.org/10.1042/bst20201171.

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Piwi-interacting RNAs (piRNAs) defend animal gonads by guiding PIWI-clade Argonaute proteins to silence transposons. The nuclear Piwi/piRNA complexes confer transcriptional repression of transposons, which is accompanied with heterochromatin formation at target loci. On the other hand, piRNA clusters, genomic loci that transcribe piRNA precursors composed of transposon fragments, are often recognized by piRNAs to define their heterochromatic identity. Therefore, Piwi/piRNA complexes must resolve this conundrum of silencing transposons while allowing the expression of piRNA precursors, at least in Drosophila germlines. This review is focused on recent advances how the piRNA pathway deals with this genetic conflict.

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Wang, Jiajia, Yirong Shi, Honghong Zhou, et al. "piRBase: integrating piRNA annotation in all aspects." Nucleic Acids Research 50, no. D1 (2021): D265—D272. http://dx.doi.org/10.1093/nar/gkab1012.

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Abstract Piwi-interacting RNAs are a type of small noncoding RNA that have various functions. piRBase is a manually curated resource focused on assisting piRNA functional analysis. piRBase release v3.0 is committed to providing more comprehensive piRNA related information. The latest release covers &gt;181 million unique piRNA sequences, including 440 datasets from 44 species. More disease-related piRNAs and piRNA targets have been collected and displayed. The regulatory relationships between piRNAs and targets have been visualized. In addition to the reuse and expansion of the content in the previous version, the latest version has additional new content, including gold standard piRNA sets, piRNA clusters, piRNA variants, splicing-junction piRNAs, and piRNA expression data. In addition, the entire web interface has been redesigned to provide a better experience for users. piRBase release v3.0 is free to access, browse, search, and download at http://bigdata.ibp.ac.cn/piRBase.

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Kofler, Robert. "piRNA Clusters Need a Minimum Size to Control Transposable Element Invasions." Genome Biology and Evolution 12, no. 5 (2020): 736–49. http://dx.doi.org/10.1093/gbe/evaa064.

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Abstract piRNA clusters are thought to repress transposable element (TE) activity in mammals and invertebrates. Here, we show that a simple population genetics model reveals a constraint on the size of piRNA clusters: The total size of the piRNA clusters of an organism must exceed 0.2% of a genome to repress TE invasions. Moreover, larger piRNA clusters accounting for up to 3% of the genome may be necessary when populations are small, transposition rates are high, and TE insertions are recessive. If piRNA clusters are too small, the load of deleterious TE insertions that accumulate during a TE invasion may drive populations extinct before an effective piRNA-based defense against the TE can be established. Our findings are solely based on three well-supported assumptions: 1) TEs multiply within genomes, 2) TEs are mostly deleterious, and 3) piRNA clusters act as transposon traps, where a single insertion in a cluster silences all TE copies in trans. Interestingly, the piRNA clusters of some species meet our observed minimum size requirements, whereas the clusters of other species do not. Species with small piRNA clusters, such as humans and mice, may experience severe fitness reductions during invasions of novel TEs, which is possibly even threatening the persistence of some populations. This work also raises the important question of how piRNA clusters evolve. We propose that the size of piRNA clusters may be at an equilibrium between evolutionary forces that act to expand and contract piRNA clusters.

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Huang, Xinya, Peng Cheng, Chenchun Weng, et al. "A chromodomain protein mediates heterochromatin-directed piRNA expression." Proceedings of the National Academy of Sciences 118, no. 27 (2021): e2103723118. http://dx.doi.org/10.1073/pnas.2103723118.

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PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.

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Ji, Qun, Zhengli Xie, Wu Gan, Lumin Wang, and Wei Song. "Identification and Characterization of PIWI-Interacting RNAs in Spinyhead Croakers (Collichthys lucidus) by Small RNA Sequencing." Fishes 7, no. 5 (2022): 297. http://dx.doi.org/10.3390/fishes7050297.

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PIWI-interacting RNAs (piRNAs) are an emerging class of small RNAs which protect the animal germline genome against deleterious transposable elements. Nevertheless, the characteristics and sex-related expression patterns of piRNA in Collichthys lucidus remain unknown. In this study, we first performed systematic next-generation high-throughput sequencing in C. lucidus ovaries and testes. We identified 3,027,834 piRNAs across six gonad libraries. Of these, 2225 piRNAs were differently expressed between testes and ovaries; 1195 were upregulated and 1030 downregulated in the testes. Interestingly, the potential target genes of 208 differentially expressed piRNAs had sex-related functions, including germ cell development, gonad development, ovarian follicle development, gamete generation, spermatid development, and spermatogenesis. Moreover, these target genes are involved in the TGF-β, Wnt, MAPK, mTOR, VEGF, and PI3K-Akt pathways. Further, 10 piRNAs were derived from Nectin2 and Mea1, which play important roles in sexual reproduction, male gamete generation, and germ cell development. We also identified 5482 piRNA clusters across the gonads, among which 139 piRNA clusters were uniquely expressed in the testes and 98 in the ovaries. The expression of core sex-related piRNA was validated by real-time PCR. Overall, our findings provide significant insights into C. lucidus’ sex-related piRNAs.

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Shoji, Keisuke, Yusuke Umemura, Susumu Katsuma, and Yukihide Tomari. "The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells." PLOS Genetics 19, no. 2 (2023): e1010632. http://dx.doi.org/10.1371/journal.pgen.1010632.

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PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many “dead” transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still “alive” and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs. (185 words)

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Geles, Konstantinos, Domenico Palumbo, Assunta Sellitto, et al. "WIND (Workflow for pIRNAs aNd beyonD): a strategy for in-depth analysis of small RNA-seq data." F1000Research 10 (May 14, 2021): 1. http://dx.doi.org/10.12688/f1000research.27868.2.

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Current bioinformatics workflows for PIWI-interacting RNA (piRNA) analysis focus primarily on germline-derived piRNAs and piRNA-clusters. Frequently, they suffer from outdated piRNA databases, questionable quantification methods, and lack of reproducibility. Often, pipelines specific to miRNA analysis are used for the piRNA research in silico. Furthermore, the absence of a well-established database for piRNA annotation, as for miRNA, leads to uniformity issues between studies and generates confusion for data analysts and biologists. For these reasons, we have developed WIND (Workflow for pIRNAs aNd beyonD), a bioinformatics workflow that addresses the crucial issue of piRNA annotation, thereby allowing a reliable analysis of small RNA sequencing data for the identification of piRNAs and other small non-coding RNAs (sncRNAs) that in the past have been incorrectly classified as piRNAs. WIND allows the creation of a comprehensive annotation track of sncRNAs combining information available in RNAcentral, with piRNA sequences from piRNABank, the first database dedicated to piRNA annotation. WIND was built with Docker containers for reproducibility and integrates widely used bioinformatics tools for sequence alignment and quantification. In addition, it includes Bioconductor packages for exploratory data and differential expression analysis. Moreover, WIND implements a "dual" approach for the evaluation of sncRNAs expression level quantifying the aligned reads to the annotated genome and carrying out an alignment-free transcript quantification using reads mapped to the transcriptome. Therefore, a broader range of piRNAs can be annotated, improving their quantification and easing the subsequent downstream analysis. WIND performance has been tested with several small RNA-seq datasets, demonstrating how our approach can be a useful and comprehensive resource to analyse piRNAs and other classes of sncRNAs.

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Geles, Konstantinos, Domenico Palumbo, Assunta Sellitto, et al. "WIND (Workflow for pIRNAs aNd beyonD): a strategy for in-depth analysis of small RNA-seq data." F1000Research 10 (July 12, 2021): 1. http://dx.doi.org/10.12688/f1000research.27868.3.

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Geles, Konstantinos, Domenico Palumbo, Assunta Sellitto, et al. "WIND (Workflow for pIRNAs aNd beyonD): a strategy for in-depth analysis of small RNA-seq data." F1000Research 10 (January 4, 2021): 1. http://dx.doi.org/10.12688/f1000research.27868.1.

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Huang, Ying, and Bowen Yu. "Structural studies of Rhino protein in piRNA biogenesis." Acta Crystallographica Section A Foundations and Advances 70, a1 (2014): C1589. http://dx.doi.org/10.1107/s2053273314084101.

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Small-RNA-guided gene regulation is a common biological process in eukaryotic cells. Animal germ cells are characterized by an intriguing small-RNA-mediated gene-silencing mechanism known as PIWI pathway. PIWI-interacting RNAs (piRNAs) are small, 21-30 nt single-stranded RNAs that associate with PIWI proteins. The function of piRNA is silencing transposon elements in germ line cells to keep the genome integrity since germ line cells are the only source for transmitting genetic information to the next generation. For a long time the biogenesis of piRNA and the mechanism of how it functions remains unclear. The biogenesis of piRNAs is quite different from that of other small-RNA pathways, which is independent of Dicer. piRNA biogenesis occurs through both primary and secondary pathway (or called ping-pong cycle). In drosophila transcripts from heterochromatic clusters are processed into primary piRNAs. A particularly fast evolving homologue of heterochromatin protein 1 (HP1) called Rhino binds to dual-strand piRNA clusters and is required for their production. But how does Rhino recognize histone H3 trimethylated on lysine 9? What's the difference between Rhino and other HP1 proteins? Here we show the crystal structure of Rhino both in apo form and complex form with H3K9me3. We observed a unique dimer interface in Rhino and a domain-swapping in conformational change. These findings provide insights into the molecular mechanism of the specificity of Rhino recognizing histone H3K9me3 and its function in piRNA biogenesis.

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Tsai, Shih-Ying, and Fu Huang. "Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription." PLOS Genetics 17, no. 2 (2021): e1009349. http://dx.doi.org/10.1371/journal.pgen.1009349.

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The piRNA pathway is a highly conserved mechanism to repress transposon activation in the germline in Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lack conventional promoters, and utilize heterochromatin- and HP1D/Rhino-dependent noncanonical mechanisms for transcription. However, information regarding the transcriptional regulation of piRNA clusters is limited. Here, we report that the Drosophila acetyltransferase Enok, which can activate transcription by acetylating H3K23, is critical for piRNA production from 54% of piRNA clusters including 42AB, the major piRNA source. Surprisingly, we found that Enok not only promotes rhino expression by acetylating H3K23, but also directly enhances transcription of piRNA clusters by facilitating Rhino recruitment. Taken together, our study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing.

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Kamenova, Saltanat, Aksholpan Sharapkhanova, Aigul Akimniyazova, et al. "piRNA and miRNA can Suppress the Expression of Multiple Sclerosis Candidate Genes." Nanomaterials 13, no. 1 (2022): 22. http://dx.doi.org/10.3390/nano13010022.

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Multiple sclerosis (MS) is a common inflammatory demyelinating disease with a high mortality rate. MS is caused by many candidate genes whose specific involvement has yet to be established. The aim of our study was to identify endogenous miRNAs and piRNAs involved in the regulation of MS candidate gene expression using bioinformatic methods. A program was used to quantify the interaction of miRNA and piRNA nucleotides with mRNA of the target genes. We used 7310 miRNAs from three databases and 40,000 piRNAs. The mRNAs of the candidate genes revealed miRNA binding sites (BSs), which were located separately or formed clusters of BSs with overlapping nucleotide sequences. The miRNAs from the studied databases were generally bound to mRNAs in different combinations, but miRNAs from only one database were bound to the mRNAs of some genes. For the first time, a direct interaction between the complete sequence of piRNA nucleotides and the nucleotides of their mRNA BSs of target genes was shown. One to several clusters of BSs of miRNA and piRNA were identified in the mRNA of ADAM17, AHI1, CD226, EOMES, EVI5, IL12B, IL2RA, KIF21B, MGAT5, MLANA, SOX8, TNFRSF1A, and ZBTB46 MS candidate genes. These piRNAs form the expression regulation system of the MS candidate genes to coordinate the synthesis of their proteins. Based on these findings, associations of miRNAs, piRNAs, and candidate genes for MS diagnosis are recommended.

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Fromm, Bastian, Juan Pablo Tosar, Felipe Aguilera, Marc R. Friedländer, Lutz Bachmann, and Andreas Hejnol. "Evolutionary Implications of the microRNA- and piRNA Complement of Lepidodermella squamata (Gastrotricha)." Non-Coding RNA 5, no. 1 (2019): 19. http://dx.doi.org/10.3390/ncrna5010019.

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Gastrotrichs—'hairy bellies’—are microscopic free-living animals inhabiting marine and freshwater habitats. Based on morphological and early molecular analyses, gastrotrichs were placed close to nematodes, but recent phylogenomic analyses have suggested their close relationship to flatworms (Platyhelminthes) within Spiralia. Small non-coding RNA data on e.g., microRNAs (miRNAs) and PIWI-interacting RNAs (piRNA) may help to resolve this long-standing question. MiRNAs are short post-transcriptional gene regulators that together with piRNAs play key roles in development. In a ‘multi-omics’ approach we here used small-RNA sequencing, available transcriptome and genomic data to unravel the miRNA- and piRNA complements along with the RNAi (RNA interference) protein machinery of Lepidodermella squamata (Gastrotricha, Chaetonotida). We identified 52 miRNA genes representing 35 highly conserved miRNA families specific to Eumetazoa, Bilateria, Protostomia, and Spiralia, respectively, with overall high similarities to platyhelminth miRNA complements. In addition, we found four large piRNA clusters that also resemble flatworm piRNAs but not those earlier described for nematodes. Congruently, transcriptomic annotation revealed that the Lepidodermella protein machinery is highly similar to flatworms, too. Taken together, miRNA, piRNA, and protein data support a close relationship of gastrotrichs and flatworms.

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Milyaeva, P. A., A. R. Lavrenov, I. V. Kuzmin, A. I. Kim, and L. N. Nefedova. "Regulation of Uni-Strand and Dual-Strand piRNA Clusters in Germ and Somatic Tissues in <i>Drosophila melanogaster</i> under Control of <i>rhino</i>." Генетика 59, no. 12 (2023): 1372–81. http://dx.doi.org/10.31857/s0016675823120056.

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Drosophila melanogaster is a common genetic object for research of RNA-interference pathways and mobile elements regulation. Nowadays taking a part in control of retrotransposon expression the system of piRNA-interfecence well studied in ovary tissues. It is strongly believed that D. melanogaster piRNA-interference is used for retrotransposon suppression only in gonads, and two distinct pathways of piRNA biogenesis exist. Both mechanisms use transcripts of piRNA-clusters (accumulations of truncated and defect mobile elements copies): from unstrand clusters in the first case and from dualstrand clusters in the second, transcribed with one or both DNA chains correspondingly. It is well-known that proper dualstrand clusters function depends on the gene rhino, while unistrand clusters are transcribed rhino-independent and transcripts are spliced. In this paper we show that rhino participates in unistrand flamenco transcripts splicing and the piRNA-interference significance for regulation of several retrotransposons not only in gonads, but in other organs.

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Altshuller, Yelena, Qun Gao, and Michael A. Frohman. "A C-Terminal Transmembrane Anchor Targets the Nuage-Localized Spermatogenic Protein Gasz to the Mitochondrial Surface." ISRN Cell Biology 2013 (July 15, 2013): 1–7. http://dx.doi.org/10.1155/2013/707930.

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Mitochondria, normally tubular and distributed throughout the cell, are instead found in spermatocytes in perinuclear clusters in close association with nuage, an amorphous organelle composed of RNA and RNA-processing proteins that generate piRNAs. piRNAs are a form of RNAi required for transposon suppression and ultimately fertility. MitoPLD, another protein required for piRNA production, is anchored to the mitochondrial surface, suggesting that the nuage, also known as intermitochondrial cement, needs to be juxtaposed there to bring MitoPLD into proximity with the remainder of the piRNA-generating machinery. However, the mechanism underlying the juxtaposition is unknown. Gasz, a multidomain protein of known function found in the nuage in vertebrates, is required for piRNA production and interacts with other nuage proteins involved in this pathway. Unexpectedly, we observed that Gasz, in nonspermatogenic mammalian cells lines, localizes to mitochondria and does so through a previously unrecognized conserved C-terminal mitochondrial targeting sequence. Moreover, in this setting, Gasz is able to recruit some of the normally nuage-localized proteins to the mitochondrial surface. Taken together, these findings suggest that Gasz is a nuage-localized protein in spermatocytes that facilitates anchoring of the nuage to the mitochondrial surface where piRNA generation takes place as a collaboration between nuage and mitochondrial-surface proteins.

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Le Thomas, Adrien, Evelyn Stuwe, Sisi Li, et al. "Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing." Genes & Development 28, no. 15 (2014): 1667–80. http://dx.doi.org/10.1101/gad.245514.114.

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Lee, SePil, Satomi Kuramochi-Miyagawa, Ippei Nagamori, and Toru Nakano. "Effects of transgene insertion loci and copy number on Dnmt3L gene silencing through antisense transgene-derived PIWI-interacting RNAs." RNA 28, no. 5 (2022): 683–96. http://dx.doi.org/10.1261/rna.078905.121.

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PIWI-interacting RNAs (piRNAs), which are germ cell-specific small RNAs, are essential for spermatogenesis. In fetal mouse germ cells, piRNAs are synthesized from sense and antisense RNAs of transposable element sequences for retrotransposon silencing. In a previous study, we reported that transgenic mice expressing antisense-Dnmt3L under the control of the Miwi2 promoter (Tg-Miwi2P-asDnmt3L) exhibited piRNA-mediated DNMT3L down-regulation. In this study, two transgene integration loci (B3 and E1) were identified on chromosome 18 of the Tg-Miwi2P-asDnmt3L mice; these loci were weak piRNA clusters. Crossbreeding was performed to obtain mice with the transgene cassette inserted into a single locus. DNMT3L was silenced and spermatogenesis was severely impaired in mice with the transgene cassette inserted at the B3 locus (Tg-B mice). In contrast, spermatogenesis in mice bearing the transgene at the E1 locus (Tg-E mice) was normal. The number of piRNAs for Dnmt3L in Tg-B mice was eightfold higher than that in Tg-E mice. Therefore, both gene silencing and impaired spermatogenesis depended on the transgene copy number rather than on the insertion loci. Additionally, the endogenous Dnmt3L promoter was not methylated in Tg mice, suggesting that Dnmt3L silencing was caused by post-transcriptional gene silencing. Based on these data, we discuss a piRNA-dependent gene silencing mechanism against novel gene insertions.

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Yamanaka, Soichiro, Mikiko C. Siomi, and Haruhiko Siomi. "piRNA clusters and open chromatin structure." Mobile DNA 5, no. 1 (2014): 22. http://dx.doi.org/10.1186/1759-8753-5-22.

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Yu, Bowen, and Ying Huang. "Rhino defines H3K9me3-marked piRNA clusters." Oncotarget 6, no. 25 (2015): 20740–41. http://dx.doi.org/10.18632/oncotarget.5178.

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Kawaoka, Shinpei, Kahori Hara, Keisuke Shoji, et al. "The comprehensive epigenome map of piRNA clusters." Nucleic Acids Research 41, no. 3 (2012): 1581–90. http://dx.doi.org/10.1093/nar/gks1275.

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Rakhmetullina, Aizhan, Aigul Akimniyazova, Togzhan Niyazova, et al. "Interactions of piRNAs with the mRNA of Candidate Genes in Esophageal Squamous Cell Carcinoma." Current Issues in Molecular Biology 45, no. 7 (2023): 6140–53. http://dx.doi.org/10.3390/cimb45070387.

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Recently, a database of human piRNAs (piwi-interacting RNAs) was created, which allows the study of the binding of many piRNAs to the mRNAs of genes involved in many diseases, including cancer. In the present work, we identified the piRNAs that can interact with candidate esophageal squamous cell carcinoma (ESCC) genes. The binding of 480 thousand piRNAs with the mRNAs of 66 candidate ESCC genes was studied. Bioinformatic studies found that piRNAs bind only to the mRNAs of nine candidate genes: AURKA, BMP7, GCOM1, ERCC1, MTHFR, SASH1, SIX4, SULT1A1, and TP53. It has been shown that piRNAs can bind to mRNA by overlapping nucleotide sequences in limited 3′UTR and 5′UTR regions called clusters of binding sites (BSs). The existence of clusters of piRNA BSs significantly reduces the proportion of the nucleotide sequences of these sites in the mRNA of target genes. Competition between piRNAs occurs for binding to the mRNA of target genes. Individual piRNAs and groups of piRNAs that have separate BSs and clusters of BSs in the mRNAs of two or more candidate genes have been identified in the mRNAs of these genes. This organization of piRNAs BSs indicates the interdependence of the expression of candidate genes through piRNAs. Significant differences in the ability of genes to interact with piRNAs prevent the side effects of piRNAs on genes with a lack of the ability to bind such piRNAs. Individual piRNAs and sets of piRNAs are proposed and recommended for the diagnosis and therapy of ESCC.

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Firsov, Sergei Yu, Karina A. Kosherova, and Dmitry V. Mukha. "Identification and functional characterization of the German cockroach, Blattella germanica, short interspersed nuclear elements." PLOS ONE 17, no. 6 (2022): e0266699. http://dx.doi.org/10.1371/journal.pone.0266699.

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In recent decades, experimental data has accumulated indicating that short interspersed nuclear elements (SINEs) can play a significant functional role in the regulation of gene expression in the host genome. In addition, molecular markers based on SINE insertion polymorphisms have been developed and are widely used for genetic differentiation of populations of eukaryotic organisms. Using routine bioinformatics analysis and publicly available genomic DNA and small RNA-seq data, we first described nine SINEs in the genome of the German cockroach, Blattella germanica. All described SINEs have tRNA promoters, and the start of their transcription begins 11 bp upstream of an “A” box of these promoters. The number of copies of the described SINEs in the B. germanica genome ranges from several copies to more than a thousand copies in a SINE-specific manner. Some of the described SINEs and their degenerate copies can be localized both in the introns of genes and loci known as piRNA clusters. piRNAs originating from piRNA clusters are shown to be mapped to seven of the nine types of SINEs described, including copies of SINEs localized in gene introns. We speculate that SINEs, localized in the introns of certain genes, may regulate the level of expression of these genes by a PIWI-related molecular mechanism.

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Kofler, Robert. "Dynamics of Transposable Element Invasions with piRNA Clusters." Molecular Biology and Evolution 36, no. 7 (2019): 1457–72. http://dx.doi.org/10.1093/molbev/msz079.

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Lipps, Northe, Figueiredo, et al. "Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA." Biomolecules 9, no. 11 (2019): 666. http://dx.doi.org/10.3390/biom9110666.

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Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell–cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Extracellular vesicles were isolated from the serum of 23 patients with chronic thromboembolic pulmonary hypertension (CTEPH) and 23 controls using two individual methods: a column-based method or by precipitation. Extracellular vesicle- associated RNAs were analyzed by next-generation sequencing applying molecular barcoding, and differentially expressed small non-coding RNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). We identified 18 microRNAs and 21 P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) or piRNA clusters that were differentially expressed in CTEPH patients compared with controls. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodeling. Expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. Thus, we identified the extracellular vesicle- derived piRNA, DQ593039, as a potential biomarker for pulmonary hypertension and right heart disease.

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Aravin, A. A., R. Sachidanandam, A. Girard, K. Fejes-Toth, and G. J. Hannon. "Developmentally Regulated piRNA Clusters Implicate MILI in Transposon Control." Science 316, no. 5825 (2007): 744–47. http://dx.doi.org/10.1126/science.1142612.

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Zhang, Fan, Jie Wang, Jia Xu, et al. "UAP56 Couples piRNA Clusters to the Perinuclear Transposon Silencing Machinery." Cell 151, no. 4 (2012): 871–84. http://dx.doi.org/10.1016/j.cell.2012.09.040.

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Lillestøl, Reidun, Peter Redder, Roger A. Garrett, and Kim Brügger. "A putative viral defence mechanism in archaeal cells." Archaea 2, no. 1 (2006): 59–72. http://dx.doi.org/10.1155/2006/542818.

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Clusters of regularly spaced direct repeats, separated by unconserved spacer sequences, are ubiquitous in archaeal chromosomes and occur in some plasmids. Some clusters constitute around 1% of chromosomal DNA. Similarly structured clusters, generally smaller, also occur in some bacterial chromosomes. Although early studies implicated these clusters in segregation/partition functions, recent evidence suggests that the spacer sequences derive from extrachromosomal elements, and, primarily, viruses. This has led to the proposal that the clusters provide a defence against viral propagation in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based) may be evolutionarily related to the interference RNA systems (siRNA and miRNA), which are common in eukarya. Here, we analyze all the current data on archaeal repeat clusters and provide some new insights into their diverse structures, transcriptional properties and mode of structural development. The results are consistent with larger cluster transcripts being processed at the centers of the repeat sequences and being further trimmed by exonucleases to yield a dominant, intracellular RNA species, which corresponds approximately to the size of a spacer. Furthermore, analysis of the extensive clusters ofSulfolobus solfataricusstrains P1 and P2B provides support for the presence of a flanking sequence adjoining a cluster being a prerequisite for the incorporation of new spacer-repeat units, which occurs between the flanking sequence and the cluster. An archaeal database summarizing the data will be maintained at http://dac.molbio.ku.dk/dbs/SRSR/.

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Babenko, Vladimir, Anton Bogomolov, Roman Babenko, Elvira Galieva, and Yuriy Orlov. "CpG islands’ clustering uncovers early development genes in the human genome." Computer Science and Information Systems 15, no. 2 (2018): 473–85. http://dx.doi.org/10.2298/csis170523004b.

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We address the problem of the annotation of CpG islands (CGIs) clusters in the human genome. Upon analyzing gene content within CGIs clusters, piRNA, tRNA, and miRNA-encoding genes were found as well as CpG-rich homeobox genes reported previously. Chromosome-wide CGI density is positively correlated with replication timing, confirming that CGIs may serve as open chromatin markers. Early embryonic stage expressed KRAB-ZNF genes abundant at chromosome 19 were found to be interlinked with CGI clusters. We detected that a number of long CGIs and CGI clusters are, in fact, tandem copies with multiple annotated macrosatellites and paralogous genes. This finding implies that tandem expansion of CGIs may serve as a substrate for nonhomologous recombination events.

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Mohamed, Mourdas, Nguyet Thi-Minh Dang, Yuki Ogyama, et al. "A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore." Cells 9, no. 8 (2020): 1776. http://dx.doi.org/10.3390/cells9081776.

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Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

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Zhou, Hao, Jiajia Liu, Wei Sun, et al. "Differences in small noncoding RNAs profile between bull X and Y sperm." PeerJ 8 (September 18, 2020): e9822. http://dx.doi.org/10.7717/peerj.9822.

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The differences in small noncoding RNAs (sncRNAs), including miRNAs, piRNAs, and tRNA-derived fragments (tsRNAs), between X and Y sperm of mammals remain unclear. Here, we employed high-throughput sequencing to systematically compare the sncRNA profiles of X and Y sperm from bulls (n = 3), which may have a wider implication for the whole mammalian class. For the comparison of miRNA profiles, we found that the abundance of bta-miR-652 and bta-miR-378 were significantly higher in X sperm, while nine miRNAs, including bta-miR-204 and bta-miR-3432a, had greater abundance in Y sperm (p < 0.05). qPCR was then used to further validate their abundances. Subsequent functional analysis revealed that their targeted genes in sperm were significantly involved in nucleosome binding and nucleosomal DNA binding. In contrast, their targeted genes in mature oocyte were significantly enriched in 11 catabolic processes, indicating that these differentially abundant miRNAs may trigger a series of catabolic processes for the catabolization of different X and Y sperm components during fertilization. Furthermore, we found that X and Y sperm showed differences in piRNA clusters distributed in the genome as well as piRNA and tsRNA abundance, two tsRNAs (tRNA-Ser-AGA and tRNA-Ser-TGA) had lower abundance in X sperm than Y sperm (p < 0.05). Overall, our work describes the different sncRNA profiles of X and Y sperm in cattle and enhances our understanding of their potential roles in the regulation of sex differences in sperm and early embryonic development.

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Asif-Laidin, Amna, Valérie Delmarre, Jeanne Laurentie, Wolfgang J. Miller, Stéphane Ronsseray, and Laure Teysset. "Short and long-term evolutionary dynamics of subtelomeric piRNA clusters in Drosophila." DNA Research 24, no. 5 (2017): 459–72. http://dx.doi.org/10.1093/dnares/dsx017.

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Akulenko, Natalia, Sergei Ryazansky, Valeriya Morgunova, et al. "Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters." RNA 24, no. 4 (2018): 574–84. http://dx.doi.org/10.1261/rna.062851.117.

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Olovnikov, I. A., and A. I. Kalmykova. "piRNA clusters as a main source of small RNAs in the animal germline." Biochemistry (Moscow) 78, no. 6 (2013): 572–84. http://dx.doi.org/10.1134/s0006297913060035.

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Chang, Timothy H., Eugenio Mattei, Ildar Gainetdinov, Cansu Colpan, Zhiping Weng, and Phillip D. Zamore. "Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster." Molecular Cell 73, no. 2 (2019): 291–303. http://dx.doi.org/10.1016/j.molcel.2018.10.038.

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Kotnova, A. P., and Yu V. Ilyin. "Comparative Analysis of the Structure of Three piRNA Clusters in the Drosophila melanogaster Genome." Molecular Biology 54, no. 3 (2020): 374–81. http://dx.doi.org/10.1134/s0026893320030085.

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Akimniyazova, Aigul, Oxana Yurikova, Anna Pyrkova, et al. "In Silico Study of piRNA Interactions with the SARS-CoV-2 Genome." International Journal of Molecular Sciences 23, no. 17 (2022): 9919. http://dx.doi.org/10.3390/ijms23179919.

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A prolonged pandemic with numerous human casualties requires a rapid search for means to control the various strains of SARS-CoV-2. Since only part of the human population is affected by coronaviruses, there are probably endogenous compounds preventing the spread of these viral pathogens. It has been shown that piRNA (PIWI-interacting RNAs) interact with the mRNA of human genes and can block protein synthesis at the stage of translation. Estimated the effects of piRNA on SARS-CoV-2 genomic RNA (gRNA) in silico. A cluster of 13 piRNA binding sites (BS) in the SARS-CoV-2 gRNA region encoding the oligopeptide was identified. The second cluster of BSs 39 piRNAs also encodes the oligopeptide. The third cluster of 24 piRNA BS encodes the oligopeptide. Twelve piRNAs were identified that strongly interact with the gRNA. Based on the identified functionally important endogenous piRNAs, synthetic piRNAs (spiRNAs) are proposed that will suppress the multiplication of the coronavirus even more strongly. These spiRNAs and selected endogenous piRNAs have little effect on human 17494 protein-coding genes, indicating a low probability of side effects. The piRNA and spiRNA selection methodology created for the control of SARS-CoV-2 (NC_045512.2) can be used to control all strains of SARS-CoV-2.

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Mohn, Fabio, Grzegorz Sienski, Dominik Handler, and Julius Brennecke. "The Rhino-Deadlock-Cutoff Complex Licenses Noncanonical Transcription of Dual-Strand piRNA Clusters in Drosophila." Cell 157, no. 6 (2014): 1364–79. http://dx.doi.org/10.1016/j.cell.2014.04.031.

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Devor, Eric J., Lingyan Huang, and Paul B. Samollow. "piRNA-like RNAs in the marsupial Monodelphis domestica identify transcription clusters and likely marsupial transposon targets." Mammalian Genome 19, no. 7-8 (2008): 581–86. http://dx.doi.org/10.1007/s00335-008-9109-x.

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Zanni, V., A. Eymery, M. Coiffet, et al. "Distribution, evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA clusters." Proceedings of the National Academy of Sciences 110, no. 49 (2013): 19842–47. http://dx.doi.org/10.1073/pnas.1313677110.

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Klattenhoff, Carla, Hualin Xi, Chengjian Li, et al. "The Drosophila HP1 Homolog Rhino Is Required for Transposon Silencing and piRNA Production by Dual-Strand Clusters." Cell 138, no. 6 (2009): 1137–49. http://dx.doi.org/10.1016/j.cell.2009.07.014.

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Milyaeva, Polina A., Inna V. Kukushkina, Alexander I. Kim, and Lidia N. Nefedova. "Stress Induced Activation of LTR Retrotransposons in the Drosophila melanogaster Genome." Life 13, no. 12 (2023): 2272. http://dx.doi.org/10.3390/life13122272.

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Background: Retrotransposons with long terminal repeats (LTR retrotransposons) are widespread in all groups of eukaryotes and are often both the cause of new mutations and the source of new sequences. Apart from their high activity in generative and differentiation-stage tissues, LTR retrotransposons also become more active in response to different stressors. The precise causes of LTR retrotransposons’ activation in response to stress, however, have not yet been thoroughly investigated. Methods: We used RT-PCR to investigate the transcriptional profile of LTR retrotransposons and piRNA clusters in response to oxidative and chronic heat stresses. We used Oxford Nanopore sequencing to investigate the genomic environment of new insertions of the retrotransposons. We used bioinformatics methods to find the stress-induced transcription factor binding sites in LTR retrotransposons. Results: We studied the transposition activity and transcription level of LTR retrotransposons in response to oxidative and chronic heat stress and assessed the contribution of various factors that can affect the increase in their expression under stress conditions: the state of the piRNA-interference system, the influence of the genomic environment on individual copies, and the presence of the stress-induced transcription factor binding sites in retrotransposon sequences. Conclusions: The main reason for the activation of LTR retrotransposons under stress conditions is the presence of transcription factor binding sites in their regulatory sequences, which are triggered in response to stress and are necessary for tissue regeneration processes. Stress-induced transposable element activation can function as a trigger mechanism, triggering multiple signal pathways and resulting in a polyvariant cell response.

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Akkouche, Abdou, Bruno Mugat, Bridlin Barckmann, et al. "Piwi Is Required during Drosophila Embryogenesis to License Dual-Strand piRNA Clusters for Transposon Repression in Adult Ovaries." Molecular Cell 66, no. 3 (2017): 411–19. http://dx.doi.org/10.1016/j.molcel.2017.03.017.

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Choi, Heejin, Zhengpin Wang, and Jurrien Dean. "Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA." PLOS Genetics 17, no. 4 (2021): e1009485. http://dx.doi.org/10.1371/journal.pgen.1009485.

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piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

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Fey, Rosalyn M., Eileen S. Chow, Barbara O. Gvakharia, Jadwiga M. Giebultowicz, and David A. Hendrix. "Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads." F1000Research 11 (December 21, 2022): 1543. http://dx.doi.org/10.12688/f1000research.124724.1.

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Background: MicroRNAs are a class of small (~22nt) endogenous RNAs that regulate target transcript expression post-transcriptionally. Previous studies characterized age-related changes in diurnal transcript expression but it is not understood how these changes are regulated, and whether they may be attributed in part to changes in microRNA expression or activity with age. Diurnal small RNA expression changes with age were not previously studied. Methods: To interrogate changes in small RNA expression with age, we collected young (5 day) and old (55 day) Drosophila melanogaster around-the-clock and performed deep sequencing on size-selected RNA from whole heads. Results: We found several microRNAs with changes in rhythmicity after aging, and we investigated microRNAs which are differentially expressed with age. We found that predicted targets of differentially expressed microRNAs have RNA-binding and transcription factor activity. We used a previously published method to identify mRNA transcripts which show evidence of microRNA targeting that is altered after aging, and found several that are involved in muscle development and maintenance. Finally, we identified novel microRNAs using the random-forest-based method miRWoods, which surprisingly also discovered transfer RNA-derived fragments. Conclusions: We showed a decrease in global microRNA expression and a corresponding increase in piRNA expression during aging. We also found an increase in rhythmicity of Drosophila small RNAs during aging, including microRNAs, piRNA clusters, and novel transfer RNA-derived fragments. To our knowledge this is the first study examining diurnal small RNA expression around the clock in young and old Drosophila, and as such it paves the way for future research on changes in small RNA regulatory molecules in the context of aging.

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