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Higher-Order Organization Principles of Pre-translational mRNPs [RIPPLiT]

ABSTRACT: We demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5' and 3' end interaction.

ORGANISM(S): Homo sapiens

PROVIDER: GSE115788 | GEO | 2018/11/14

REPOSITORIES: GEO

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Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways

Project description:Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.

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mRNA composition of IRP2 mRNPs in mouse tissues

Project description:Affymetrix microarrays were used to determine the mRNA composition of mRNPs obtained by immunoprecipitation with IRP2 (iron regulatory protein 2).

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Weak interactions in higher-order chromatin organization.

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RNA content of yeast nuclear mRNPs

Project description:We isolated nuclear mRNPs from yeast using an endogenous bi-molecular tagging approach on Sub2 and Hpr1. To validate the nature of these isolated particles, extracted RNA was sequenced and differential enrichment, as well as metagene coverage analysis were performed.

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Global Analysis of Yeast mRNPs

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