Tag Archives: HTS

Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis.

Publication
MILI_MIWI

Full Text
Nat Struct Mol Biol. 19(8):773-81

Abstract
Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA-sequencing (RNA-seq) to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.

Notes

Mitochondrial protein BmPAPI modulates the length of mature piRNAs.

Publication
Papi

Full Text
RNA 19(10):1405-18

Abstract
PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3′-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.

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Identification of in vivo, conserved, TAF15 RNA binding sites reveals the impact of TAF15 on the neuronal transcriptome.

Publication
TAF15

Full Text
Cell Rep.
3(2):301-8

Abstract
RNA binding proteins (RBPs) have emerged as major causative agents of amyotrophic lateral sclerosis (ALS). To investigate the function of TAF15, an RBP recently implicated in ALS, we explored its target RNA repertoire in normal human brain and mouse neurons. Coupling high-throughput sequencing of immunoprecipitated and crosslinked RNA with RNA sequencing and TAF15 knockdowns, we identified conserved TAF15 RNA targets and assessed the impact of TAF15 on the neuronal transcriptome. We describe a role of TAF15 in the regulation of splicing for a set of neuronal RNAs encoding proteins with essential roles in synaptic activities. We find that TAF15 is required for a critical alternative splicing event of the zeta-1 subunit of the glutamate N-methyl-D-aspartate receptor (Grin1) that controls the activity and trafficking of NR1. Our study uncovers neuronal RNA networks impacted by TAF15 and sets the stage for investigating the role of TAF15 in ALS pathogenesis.

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FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns.

Publication
FUS

Full Text
RNA 19(4):498-509.

Abstract
Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS-CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts.

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A MicroRNA precursor surveillance system in quality control of MicroRNA synthesis.

Publication

microrna biogenesis img

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Mol Cell. 55(6):868-79.

Abstract
MicroRNAs (miRNAs) are essential for regulation of gene expression. Though numerous miRNAs have been identified by high-throughput sequencing, few precursor miRNAs (pre-miRNAs) are experimentally validated. Here we report a strategy for constructing high-throughput sequencing libraries enriched for full-length pre-miRNAs. We find widespread and extensive uridylation of Argonaute (Ago)-bound pre-miRNAs, which is primarily catalyzed by two terminal uridylyltransferases: TUT7 and TUT4. Uridylation by TUT7/4 not only polishes pre-miRNA 3′ ends, but also facilitates their degradation by the exosome, preventing clogging of Ago with defective species. We show that the exosome exploits distinct substrate preferences of DIS3 and RRP6, its two catalytic subunits, to distinguish productive from defective pre-miRNAs. Furthermore, we identify a positive feedback loop formed by the exosome and TUT7/4 in triggering uridylation and degradation of Ago-bound pre-miRNAs. Our study reveals a pre-miRNA surveillance system that comprises TUT7, TUT4, and the exosome in quality control of miRNA synthesis.

Notes

The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

Publication
MOV10L1

Full Text
Genes Dev. 2015 Mar 15;29(6):617-29.

Abstract
Piwi-piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5′-to-3′ directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.

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CLIP Seq Tools

GitHub link
CPAN link

Summary

CLIPSeqTools is a collection of command line applications used for the analysis of CLIP-Seq (UV cross-linking and immunoprecipitation with high-throughput sequencing) data. It offers a wide range of analyses (eg. genome read coverage, motif enrichment, relative positioning of reads of two libraries, etc). The toolbox is primarily oriented for bioinformaticians but the commands are simple enough for non experts to use.

GenOO

GenOO: A Modern Perl Framework for High Throughput Sequencing analysis

GitHub link
Full Text Publication BioArxiv

Summary

GenOO [jee-noo] is an open-source; object-oriented Perl framework specifically developed for the design of High Throughput Sequencing (HTS) analysis tools. The primary aim of GenOO is to make simple HTS analyses easy and complicated analyses possible. GenOO models biological entities into Perl objects and provides relevant attributes and methods that allow for the manipulation of high throughput sequencing data. Using GenOO as a core development module reduces the overhead and complexity of managing the data and the biological entities at hand. GenOO has been designed to be flexible, easily extendable with modular structure and minimal requirements for external tools and libraries.

Focus

  • Organize biological entities as perl objects (genomic regions, genes, transcripts, introns/exons, etc)
  • Organize sequencing entities as perl objects/attributes (sequencing reads, alignments, etc)
  • Make I/O from widely used file formats easy (SAM, BED, FASTA, FASTQ)
  • Be consistent and easily extendable

We want to keep this framework focused on the real issues found in sequencing analyses and balance being easily extendable with being focused and efficient.