HP1γ binding pre-mRNA at intronic repeats increases splicing fidelity and regulates alternative exon usage

Abstract

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1g/Cbx3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA binding activity of HP1g in transcriptionally active chromatin, we have captured and analyzed RNAs associated with this protein. We find that HP1g specifically recognizes hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1g RNA binding activity tethers unspliced pre-mRNA to chromatin via the intronic region and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and cotranscriptional splicing.showing that its role in mRNA maturation, alike its role in heterochromatin structuring, relies on its ability to associate with repeat-encoded RNAs. RESULTS HP1g associates with chromatin-enriched RNA.To better understand the relationship between HP1g and RNA, beyond its classical role in heterochromatin formation, we used a genome-wide approach to assay the interactions between HP1g and RNA on chromatin (RNAchIP; Fig. 1a). For this, we used a modification of our previously described strategy to solubilize native chromatin and produce chromatinenriched RNA fragments suitable for immunoprecipitation 25 (Supplementary Fig. 1d). HP1g -/-(KO cells) mouse embryonic fibroblast (MEF)-derived cell lines, re-complemented with FLAG-tagged HP1g (HP1g cells) as previously described were used in these assays 16 . These cells expressed ectopic FLAG-tagged HP1g at the same level as the endogenous HP1g in WT MEFs ( Supplementary Fig. 1a). As in our previous studies, the cells were treated or not with the phorbol ester PMA, an activator of the PKC signaling pathway, in order to observe the cellular response to stress on chromatin and transcription. Nuclei were isolated to obtain a chromatin-enriched RNA fraction, ( Supplementary Fig. 1a and b). Our procedure was modified to include a limited cross-linking step in order to stabilize association of HP1g with RNA, together with stringent immunoprecipitation conditions to favor direct protein-RNA associations. Under these conditions, anti-FLAG antibody did not precipitate any of the HP1g-interacting proteins we tested (HP1a, H3, RNA polymerase II) ( Supplementary Fig. 1b). When quantified by RT-qPCR, HP1g was found to associate with chromatin-enriched RNA from the Fosl1 locus with a 20-to 25-fold enrichment compared to KO cells. IP in KO cells consistently showed very low to undetectable RNA levels ( Supplementary Fig. 1c).Genome-wide analysis of the RNAchIP by HP1g (IP) versus RNA detected in the chromatin fraction (input) was then performed using Illumina sequencing on biological triplicates. Reads were then mapped onto the mouse genome. In a first approach, RNAchIPseq dat...

RNA binding proteins in osteoarthritis

“…RBPs are a series of proteins which could bind with double or singlestranded RNA and thereby demonstrate the function to influence the RNA fate by forming ribonucleoprotein (Glisovic et al, 2008;Bohnsack and Bohnsack, 2019). The main role of RBPs is RNA metabolism regulation, including mRNA stability (Shi et al, 2017), mRNA splicing (Rachez et al, 2021), translocation (Tolino et al, 2012), translation (Moore and von Lindern, 2018) as well as the corporation with noncoding RNAs (Ciafre and Galardi, 2013;Qin et al, 2020). The functions of RBPs are highly dependent on their structural features which is the RNA binding domains, and the most common domains are including RNA recognition motif (RRM), arginine-glycineglycine (RGG) motif, the hnRNP-K homology domain (KH), cold-shock domain (CSD), double-stranded RBD (dsRBD), tyrosine rich domain and the zinc finger domain (ZF) (Maris et al, 2005;Dominguez et al, 2018;Qin et al, 2020;Dobrev et al, 2021).…”