Analysis of large-scale sequencing of small RNAs

The advent of large-scale sequencing has opened up new areas of research, such as the study of Piwi-interacting small RNAs (piRNAs). piRNAs are longer than miRNAs, close to 30 nucleotides in length, involved in various functions, such as the suppression of transposons in germline. Since a large number of them (many tens of thousands) are generated from a wide range of positions in the genome, large-scale sequencing is the only way to study them. The key to understanding their genesis and biological roles is efficient analysis, which is complicated by the large volumes of sequence data. Taking account of the underlying biology is also important. We describe here novel analyses techniques and tools applied to small RNAs from germ cells in D. melanogaster, that allowed us to infer mechanism and biological function

Bacterial Argonaute Samples the Transcriptome to Identify Foreign DNA

Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea, their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15–19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22–24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid-derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids

Geoseq: a tool for dissecting deep-sequencing datasets

Gurtowski J, Cancio A, Shah H, et al. Geoseq: a tool for dissecting deep-sequencing datasets. BMC Bioinformatics. 2010;11(1): 506.Background Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Results Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Conclusions Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool

Conserved generation of short products at piRNA loci

ABSTRACT: BACKGROUND: The piRNA pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. The Piwi protein-associated small RNAs (piRNAs) guide Piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. Cleavage of the retrotransposon transcript defines at the same time the 5` end of a secondary piRNA that will in turn guide a Piwi protein to a primary piRNA precursor, thereby amplifying primary piRNAs. Although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary piRNAs are not entirely clear. RESULTS: Here we analyzed small RNAs from three mammalian species, seeking to gain further insight into the mechanisms responsible for the piRNA amplification loop. We found that in all these species piRNA-directed targeting is accompanied by the generation of short sequences that have a very precisely defined length, 19 nucleotides, and a specific spatial relationship with the guide piRNAs. CONCLUSIONS: This suggests that the processing of the 5` product of piRNA-guided cleavage occurs while the piRNA target is engaged by the Piwi protein. Although they are not stabilized through methylation of their 3` ends, the 19-mers are abundant not only in testes lysates but also in immunoprecipitates of Miwi and Mili proteins. They will enable more accurate identification of piRNA loci in deep sequencing data sets

MORC1 represses transposable elements in the mouse male germline

The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA-methylated and silent genes. ​MORC1-deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that ​MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homologue ​DNMT3L. ​Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify ​MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation

Exonuclease Domain-Containing 1 Enhances MIWI2 piRNA Biogenesis via Its Interaction with TDRD12

PIWI proteins and their associated small RNAs, called PIWI-interacting RNAs (piRNAs), restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most MILI piRNAs are derived via a slicer-independent pathway, MILI slicing loads MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1/;Tdrd12+/), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, leading to infertility. Thus, EXD1 enhances MIWI2 piRNA biogenesis via a functional interaction with TDRD12.<br /

Computational methods for analyzing small RNAs and their interaction partners with large-scale techniques

This thesis describes the computational tools and analyzes developed to characterize small regulatory RNAs and their interaction partners using large-scale techniques. Following an introduction into the emerging world of small regulatory RNAs, our methodology for analyzing small RNAs from deep-sequencing data is described (chapter 2). This methodology allows the classification of small RNAs obtained by sequencing and provides several downstream analysis tools such as expression profiling and miRNA sample comparison. It has been integrated with a miRNA target prediction method into a web server which allows one to explore tissue-specific miRNA targeting (chapter 3). In the fourth chapter, an experimental procedure for genome-wide identification of miRNA targets is outlined. With this procedure, we identified the mRNAs, that are targeted by the most abundant miRNAs in HEK293 cells. Importantly, the experimental protocol enabled us to identify the exact location of the miRNA-mRNA interaction site within the mRNA as well as the precise position of the mRNA-protein crosslink. The fifth and sixth chapter describe our studies of murine embryonic stem cells and oocytes that are devoid of Dicer. The murine specific miR-290 cluster has been identified as an important regulator in embryonic stem cells. The loss of pluripotency in Dicer-/- embryonic stem cells has been linked to primary and secondary targets of the miR-290 cluster. In contrast, our analysis of Dicer-/- oocytes revealed that the miRNA pathway plays only a minor part during oocyte maturation, and loss of Dicer affects mainly the endo-siRNA pathway. Finally, we reanalyzed piRNA sequence reads from various species (chapter 6). This analysis revealed an unexpected 19 nt long processing product which is generated during piRNA biogenesis

Regulation of Gene Expression by Non-coding RNAs in Arabidopsis and Brachypodium

Title from PDF of title page, viewed on January 14, 2016Dissertation advisor: Julia ChekanovaVitaIncludes bibliographical references (pages 200-238)Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2014Expansion of high-throughput sequencing technology has increased our understanding in the importance of pervasive transcription and the resulting indispensable molecules, noncoding RNAs (ncRNAs). A significant effort has been made in determining the biological relevance of ncRNAs, including the small RNAs (smRNAs) and long ncRNAs (lncRNAs). Studies have shown that ncRNAs can have very important regulatory functions such as the establishment and maintenance of the epigenetic architecture of eukaryotic genomes. However, these studies have only revealed the surface of ncRNAs’ functions in cells. Motivated by the previously “hidden” transcriptome regulated by the Arabidopsis exosome, we examined the role of ncRNAs in regulation of gene expression in two plant model systems, Arabidopsis and Brachypodium. We set out to investigate whether the Arabidopsis exosome complex modulates gene expression through regulating ncRNAs, including both smRNAs and lncRNAs. Thus, the role of Arabidopsis exosome complex in regulating smRNA metabolism was extensively investigated and we revealed the differences between Arabidopsis core exosome, yeast and human exosomes in modulating smRNAs. Although we have only begun to categorize the lncRNAs regulated by the Arabidopsis exosome, our data so far has led to intriguing speculations, including the possible role of exosome in transcriptional regulation via controlling lncRNAs. The challenges still remain, including establishing the regulatory role of specific lncRNAs regulated by the exosome and other factors at transcriptional or post-transcriptional levels in different cellular contexts, and most importantly, how the interaction between these lncRNAs and the chromatin is mediated. We also investigated the role of smRNAs in biotic and abiotic stress responses in Brachypodium. We identified a group of endogenous, stress-induced small interfering RNAs, sutr-siRNAs, and its novel mechanism in targeting cis elements involved in splice site selection. This finding will stimulate research of smRNA mediated stress responses in agricultural important crops. It also provides a framework for more suitable experimental systems, such as the Drosophila and mammalian cell cultures, to further investigate the associated mechanistic details of sutr-siRNAs. In this dissertation, we revealed new insights and detailed molecular mechanisms of the Arabidopsis exosome complex in regulation of gene expression, as well as provided a novel regulatory mechanism of endogenous siRNAs by being involved in splicing. Although many challenges still remain to fully determine the mechanistic details and biological significance of plant ncRNAs, the work presented in this dissertation has added to our understanding in regulation of gene expression through ncRNAs in plants.Introduction -- The role of the arabidopsis exosome in SiRNA-independent silencing of heterochromatic loci -- Characterization of the sub-group of SmRNAs producing genomic loci affected by the defect in arabidopsis exosome complex- Stress-induced endogenous SiRNAs targeting regulatory intron sequences in brachypodium -- Conclusion and future directions -- Appendix I. List of associated publications -- Appendix II. curriculum vita