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Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [sgRNA screen]

ABSTRACT: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [sgRNA screen]

PROVIDER: PRJNA886717 | ENA |

REPOSITORIES: ENA

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Convergence of distinct RNA-silencing pathways on GW182/Tnrc6

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Project description:The RNA-binding protein Trim71/Lin41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development and cancer. Trim71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Deposited data are mass spectrometry data from immunoprecipitation experiments with endogenously tagged Trim71, Ago2, and Tnrc6a. Experiments are carried out in mouse embryonic stem cells (mESCs) and uncover reciprocal interactions of Trim71, Ago2, and Tnrc6a. Trim71 protein interactions are largely independent of Ago2 levels, but strongly depend on the presence of RNA. A second set of experiments is mass spectrometry data from immunoprecipitation experiments in mESCs overexpressing a tagged peptide derived from Homo sapiens TNRC6B (‘FLAG-HA::T6BWT::Cherry’), that is known to block the Tnrc6-Ago2 interaction. We show that a wild-type version strongly binds Ago1 and Ago2, but not Trim71, while a mutant version binds neither Ago1, 2, nor Trim71.

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Project description:This SuperSeries is composed of the SubSeries listed below.

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Impact of TNRC6 isoforms on gene expression and splicing

Project description:Through the RNA interference (RNAi) pathway, small RNAs can influence translation, splicing, transcriptional activation, and transcriptional repression. RNAi is carried out by the targeting Argonaute (Ago) effector protein and the facilitating TNRC6 (also known as GW182) scaffolding protein. Here, we use a suite of gene knockout cell lines and high-throughput sequencing to gather definitive answers about the TNRC6 paralogs, their roles in the cell, and their relation to Ago knockout cell lines. Each subsequent TNRC6 paralog knockout caused more gene changes and few genes overlapped between the single TNRC6 paralog knockouts. In addition, TNRC6 knockout and Ago knockout cell lines’ gene expression profiles are highly correlated with one another, indicating the important regulatory functions these proteins share. Overall we found that in spite of less than 40% sequence identity, TNRC6 paralogs are functionally redundant and can replace one another for core RNAi functions.

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