Argonaute binding within human nuclear RNA and its impact on alternative splicing

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 (RNAseq) 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 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 were 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 a natural regulatory mechanism, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction

“…We isolated nuclear and cytoplasmic fractions from each population and confirmed AGO2 protein localization in both the nucleus and cytoplasm of meiotic and postmeiotic male germ cells, in agreement with previous observations in other mammalian tissues ( Fig. 1 A; Kim et al 2011 ; Gagnon et al 2014 ; Ravid et al 2020 ; Chu et al 2021 ), suggesting that AGO2 can function in both the nucleus and cytoplasm at multiple steps of spermatogenic development.…”

“…Unlike cytoplasmic AGO2, which primarily binds to 3′ UTRs of target transcripts ( Chi et al 2009 ; Hafner et al 2010 ), most nuclear AGO2 binding sites were located in intronic regions of unspliced transcripts (62% of CLIP peaks in meiotic cells and 74% of CLIP peaks in postmeiotic cells), consistent with a recent study in a human cancer cell line ( Fig. 2 E; Chu et al 2021 ). Most nuclear RNA targets corresponded to protein-coding genes in both meiotic and postmeiotic cells ( Fig.…”

Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

“…2I). Although previous AGO2 CLIP and chimeric CLIP studies have sometimes yielded a significant number of peaks in introns or linked to rRNA, tRNA, or other non-coding RNAs (Chu et al, 2021; Helwak et al ., 2013), the particular emergence of non-coding signal only in no-gel experiments here suggests that it likely represents false positive signal. Previous CLASH studies utilizing spike-ins of E. coli RNA and Drosophila S2 cells (Moore et al ., 2015) or yeast lysate (Helwak et al ., 2013) into human cell lysate estimated a low (1-5%) rate of interactions formed in vitro after cell lysis.…”

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

“…This suggested AGO1 works with FCA to promote proximal COOLAIR . Argonaute proteins have been documented to influence alternative splicing in other organisms ( 17 – 19 , 34 ). We therefore hypothesized the shift to distal COOLAIR in ago1 mutant might be due to a shift in use of the distal splice acceptor site.…”