Monitored eCLIP: high accuracy mapping of RNA-protein interactions

International audienceCLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotiny-lated linker is ligated at the 5 end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment

Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms

Insights into the design and interpretation of iCLIP experiments

Abstract Background Ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) identifies the sites on RNAs that are in direct contact with RNA-binding proteins (RBPs). Several variants of CLIP exist, which require different computational approaches for analysis. This variety of approaches can create challenges for a novice user and can hamper insights from multi-study comparisons. Here, we produce data with multiple variants of CLIP and evaluate the data with various computational methods to better understand their suitability. Results We perform experiments for PTBP1 and eIF4A3 using individual-nucleotide resolution CLIP (iCLIP), employing either UV-C or photoactivatable 4-thiouridine (4SU) combined with UV-A crosslinking and compare the results with published data. As previously noted, the positions of complementary DNA (cDNA)-starts depend on cDNA length in several iCLIP experiments and we now find that this is caused by constrained cDNA-ends, which can result from the sequence and structure constraints of RNA fragmentation. These constraints are overcome when fragmentation by RNase I is efficient and when a broad cDNA size range is obtained. Our study also shows that if RNase does not efficiently cut within the binding sites, the original CLIP method is less capable of identifying the longer binding sites of RBPs. In contrast, we show that a broad size range of cDNAs in iCLIP allows the cDNA-starts to efficiently delineate the complete RNA-binding sites. Conclusions We demonstrate the advantage of iCLIP and related methods that can amplify cDNAs that truncate at crosslink sites and we show that computational analyses based on cDNAs-starts are appropriate for such methods

Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains

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Splicing enhances translation in mammalian cells: an additional function of the exon junction complex

In mammalian cells, spliced mRNAs yield greater quantities of protein per mRNA molecule than do otherwise identical mRNAs not made by splicing. This increased translational yield correlates with enhanced cytoplasmic polysome association of spliced mRNAs, and is attributable to deposition of exon junction complexes (EJCs). Translational stimulation can be replicated by tethering the EJC proteins Y14, Magoh, and RNPS1 or the nonsense-mediated decay (NMD) factors Upf1, Upf2, and Upf3b to an intronless reporter mRNA. Thus, in addition to its previously characterized role in NMD, the EJC also promotes mRNA polysome association. Furthermore, the ability to stimulate translation when bound inside an open reading frame appears to be a general feature of factors required for NMD

Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities

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The exon–exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. When assembled in vitro, this so-called ‘exon–exon junction complex’ (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm

5′-End RET Splicing: Absence of Variants in Normal Tissues and Intron Retention in Pheochromocytomas

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