1. Argonaute binding within human nuclear RNA and its impact on alternative splicing.
- Author
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Chu Y, Yokota S, Liu J, Kilikevicius A, Johnson KC, and Corey DR
- Subjects
- Argonaute Proteins deficiency, Base Pairing, Base Sequence, Binding Sites, Cell Nucleus genetics, Cell Nucleus metabolism, Cytoplasm genetics, Cytoplasm metabolism, Eukaryotic Initiation Factors deficiency, Exons, HCT116 Cells, Humans, Immunoprecipitation, Introns, MicroRNAs antagonists & inhibitors, MicroRNAs metabolism, Oligoribonucleotides genetics, Oligoribonucleotides metabolism, Protein Binding, RNA, Nuclear metabolism, Sequence Analysis, RNA, Alternative Splicing, Argonaute Proteins genetics, Eukaryotic Initiation Factors genetics, MicroRNAs genetics, RNA, Nuclear genetics
- Abstract
Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease., (© 2021 Chu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.) more...
- Published
- 2021
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