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Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]

ABSTRACT: Germ cells employ elaborate mechanisms to maintain and protect their genetic material, and also to regulate gene expression during the complex differentiation process of gametogenesis. Piwi proteins, a subclade of the Argonaute family, are expressed mainly in the germline and bind piRNAs, a novel and diverse class of small RNAs whose biogenesis and putative functions are still largely elusive. We employed High Throughput Sequencing after Crosslinking and Immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili and Miwi, two mouse Piwi proteins. Our analysis outlines a model for primary piRNA biogenesis in postnatal mouse and indicates that piRNAs do not mediate target RNA recognition, but rather are the end products of RNA processing. Moreover, we identify a set of mRNAs essential for spermiogenesis that are bound and regulated by Miwi, directly implicating Piwi proteins in the control of gene expression at key time points of spermiogenesis. HITS-CLIP (High Throughput Sequencing after Crosslinking and Immunoprecipitation) experiments targeting two mouse Piwi proteins Mili and Miwi.

ORGANISM(S): Mus musculus

SUBMITTER: Qi Zheng

PROVIDER: E-GEOD-27622 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]

Project description:Germ cells employ elaborate mechanisms to maintain and protect their genetic material, and also to regulate gene expression during the complex differentiation process of gametogenesis. Piwi proteins, a subclade of the Argonaute family, are expressed mainly in the germline and bind piRNAs, a novel and diverse class of small RNAs whose biogenesis and putative functions are still largely elusive. We employed High Throughput Sequencing after Crosslinking and Immunoprecipitation (HITS-CLIP) coupled with RNA-Seq to characterize the genome-wide target RNA repertoire of Mili and Miwi, two mouse Piwi proteins. Our analysis outlines a model for primary piRNA biogenesis in postnatal mouse and indicates that piRNAs do not mediate target RNA recognition, but rather are the end products of RNA processing. Moreover, we identify a set of mRNAs essential for spermiogenesis that are bound and regulated by Miwi, directly implicating Piwi proteins in the control of gene expression at key time points of spermiogenesis.

2012-07-20 | GSE27622 | GEO

Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [RNA-Seq]

2012-07-20 | GSE27609 | GEO

MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes

Project description:The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation. CLIP-Seq (Crosslinking Immunoprecipitation coupled with high-throughput sequencing) experiments targeting Miwi in isolated round spermatids from mouse testis.

2015-04-09 | E-GEOD-67683 | biostudies-arrayexpress

Mili-IP, Miwi-IP, and total RNA from mouse adult testis

Project description:We deep-sequenced small RNAs after immunoprecipitation of Mili or Miwi, as well as total small RNA from adult mouse testis. The goal of this experiment is to more deeply characterize the piRNA pool from adult mouse testes, using the Illumina platform.

2010-01-04 | GSE19172 | GEO

Mili-IP, Miwi-IP, and total RNA from mouse adult testis

2010-01-04 | E-GEOD-19172 | biostudies-arrayexpress

Ribosome profiling study of Miwi-null testes

Project description:piRNAs are a novel class of small noncoding RNAs that were recently identified in animal germ cells. This class of small noncoding RNAs is specifically associated with an evolutionarily conserved PIWI family proteins, which belong to the germline-specific members of the Argonaute protein family and are indispensable to germline development in animals. The PIWI/piRNA pathway has been deemed as an innate immune system that prevents mobile genetic elements from destabilizing DNA and that protects genome integrity in animal germ cells. By ribosome profiling using Miwi-null testes, we identified a group of ~600 mRNAs as likely direct piRNA targets for translational regulation in mouse spermatids. These results suggest that the mouse PIWI (MIWI)/piRNA is responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into elongated spermatids.

2019-12-15 | GSE139399 | GEO

Mus musculus

Project description:Target RNA repertoire of mouse Mili and Miwi proteins reveals piRNA biogenesis and Miwi function in spermiogenesis [HITS-CLIP]

| PRJNA141769 | ENA

Genome-wide double-stranded RNA sequencing reveals the functional significance of base-paired RNAs in Arabidopsis

Project description:The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation requires formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we use high-throughput sequencing to interrogate all base-paired RNA in Arabidopsis thaliana, and identify ~200 new small (sm)RNA-producing substrates of RNA-DEPENDENT RNA POLYMERASE 6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5’ and 3’ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. These findings highlight the importance of base-paired RNAs in eukaryotes, and present an approach that should be widely applicable for the analysis of this key structural feature of RNA. Double-stranded (dsRNA) specific RNA sequencing (dsRNA-seq) in the unopened flowerbuds of wild-type col-0 plants and rdr6 mutant plants, including 2 samples of col-0 dsRNA (1X- or 2X- Ribominus-treated samples) and 1 sample of rdr6 dsRNA. Corresponding two smRNA libraries (smRNA-seq) of both wild-type col-0 plants and rdr6 mutant plants of the same tissue are also presented.

2010-10-04 | E-GEOD-23439 | biostudies-arrayexpress

Multiple epigenetic mechanisms and the piRNA pathway enforce LINE1 silencing during adult spermatogenesis

Project description:The Piwi protein Mili and the Piwi-interacting RNA (piRNA) pathway are required to establish L1 DNA methylation during epigenetic reprogramming that accompanies male germ cell development. Although Mili is expressed throughout most of adult spermatogenesis, its function therein remains unknown. We find that Mili displays a hitherto unappreciated dynamic expression profile during spermatogenesis, expressed in the mitotic spermatogonia but absent at the early meiotic stages only to reappear in late zygotene spermatocytes. Here we show using conditional mutagenesis that Mili's endonuclease activity post-transcriptionally cleaves L1 transcripts commencing upon its reappearance in late zygotene spermatocytes even in the presence of normal L1 DNA methylation

2013-07-03 | E-MTAB-1557 | biostudies-arrayexpress

Global analysis of RNA secondary structure conservation between two metazoans

Project description:The secondary structure of RNA is necessary for its maturation, regulation, and processing. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we identify evolutionarily conserved features of RNA secondary structure in metazoans by applying our high-throughput, sequencing-based, structure-mapping approach to Drosophila melanogaster and Caenorhabditis elegans. This analysis reveals key structural patterns across protein-coding transcripts that indicate RNA folding is influential to protein translation and microRNA-mediated targeting and/or regulation of mRNAs in animals. Additionally, we uncover a novel population of highly base-paired RNAs, many of which are likely functional, long, non-coding RNAs. Finally, we identify and characterize ~180 structural motifs of mRNAs that are under positive or negative selection in these metazoans, thereby revealing a large set of RNA structures that are likely functional. Overall, our findings highlight the significance of secondary structure within RNA molecules, and provide the first comprehensive evidence of widespread RNA secondary structure conservation in animals. Double-stranded (dsRNA) specific RNA sequencing (dsRNA-seq) and single-stranded (ssRNA) specific RNA sequencing (ssRNA-seq) in Drosophila DL1 cells and C. elegans mixed stage N2 worms. Each of the four samples (dsRNA/Dmel, ssRNA/Dmel, dsRNA/Cel, and ssRNA/Cel) was sequenced separately on both Illumina GA-IIx and Hiseq2000, giving a total of eight datasets. Two corresponding smRNA libraries (smRNA-seq) of the same DL1 cells and mixed stage N2 worms are also presented.

2012-01-26 | E-GEOD-29571 | biostudies-arrayexpress

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