Ribosome profiling study of Miwi-null testes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA579549 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10347710_1.fastq.gz | Fastqsanger.gz | |||
| SRR10347710_2.fastq.gz | Fastqsanger.gz | |||
| SRR10347711_1.fastq.gz | Fastqsanger.gz | |||
| SRR10347711_2.fastq.gz | Fastqsanger.gz | |||
| SRR10347712_1.fastq.gz | Fastqsanger.gz |
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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
Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs. piRNA sequencing, RNA immunoprecipitation, and expression measurements (RNA-Seq and ribosome profiling) in wild-type and Mael -/- testes
2014-07-25 | E-GEOD-50983 | biostudies-arrayexpress
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.
2015-04-09 | GSE67683 | GEO
Project description:Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs.
2014-07-25 | GSE50983 | GEO
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
Project description:The null pacman allele pcm[14] causes at lethaility pupariation. Lethality can be rescued by driving expression of wild-type pacman in somatic cells during development using the UAS-GAL4 system. Use of the driver arm-GAL4 allows adult flies to develop, but pacman is not expressed in the germline cells. RNA was prepared from null pacman testes from rescued flies, parental controls (UAS line and GAL4 line) and from a genetic background control for pcm[14] (a line generated by a neutral excision of the P-element used to induce the pcm[14] deletion). Total RNA was prepared from adult Drosophila testes. Sequencing of each genotype was replicated 3 times with a read depth of 12-15 million reads per replicate.
2020-06-01 | E-MTAB-4515 | biostudies-arrayexpress
Project description:Gene expression profiles of null Pacman/Xrn1 mutant Drosophila testes
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Project description:We reasoned that the pi-RISC, by virtue of the enormous sequence portfolio of piRNAs, might mediate elimination of a large variety of mRNAs in late stages of spermiogenesis. Both Miwi-null and Caf1-null mice showed early spermiogenic arrest, preventing us from determining the role of MIWI and CAF1 in elongating spermatids using the existing genetic models. We therefore used microarrays to detail the global effect of MIWI or CAF1 on mRNA levels in mouse elongating spermatids. Using GFP+ elongating spermatids sorted from mouse testes transduced with shMiwi:GFP, shCaf1:GFP, or control pSilencer:GFP, we performed transcriptome profiling on Affymetrix mouse arrays.
2014-07-11 | E-GEOD-59291 | biostudies-arrayexpress
Project description:ABCE1/Rli1 functions as a ribosome recycling factor in vitro, but has not been shown to be crucial for recycling in vivo. We use ribosome profiling and biochemistry to define the role of Rli1 in living yeast. When Rli1 levels were diminished, 80S ribosomes accumulated at stop codons and, surprisingly, in the adjoining 3'UTRs of most genes. While ribosomes did not show preference for any reading frame in the 3'UTR, their enrichment at stop codons and His codons after histidine starvation is consistent with aberrant 3'UTR translation. Predicted 3'UTR translation products were detected by Western analysis and mass spectrometry, and their small sizes indicate a reinitiation mechanism. Eliminating ribosome-rescue factor Dom34 dramatically increases 3'UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and for overall gene expression as it controls ribosome homeostasis and 3'UTR translation.
2015-06-02 | GSE69414 | GEO
Project description:Animal germlines express small RNAs called PIWI-interacting RNAs (piRNAs) that guide their associated PIWI endoribonucleases to destroy transposon transcripts, ensuring animal fertility. Mouse MIWI binds millions of sequences called pachytene piRNAs that use partial complementarity to target transposons and cellular mRNAs. Here we report the first cryo-EM structure of the MIWI-pachytene piRNA complex isolated from mouse testes. It shows a positively charged channel where the piRNA is held, with specific recognition of the signature first nucleotide uridine being mediated by amino acids within the MID and PIWI domains. The first six nucleotides of the guide RNA are visible and take up the A-form conformation to facilitate pairing with the target. Importantly, part of the channel accommodating the seed sequence is wider than that for insect PIWI, likely explaining the reported tolerance of MIWI to seed sequence mismatches. The catalytic site in the PIWI endonuclease domain presents an inactive state, with the loop containing one of the catalytic residues (E671) requiring structural re-orientation to adopt the catalytically competent state. Finally, we identify a conserved pre-formed pocket on the PIWI domain that may serve to accommodate a conserved tryptophan from the interacting factor GTSF1. Modelling shows how this interaction with GTSF1 can allosterically enforce reorganization of the catalytic pocket to promote small RNA-guided endoribonuclease activity. Our structure informs how MIWI functions using relaxed target engagement rules and associates with its interacting partner.
2026-01-13 | GSE304875 | GEO