High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

Abstract

ABSTRACThnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.

hnRNPs Interacting with mRNA Localization Motifs Define AxoNAl RNA Regulons

“…Comparison of these RBP-mRNA interactomes showed highest overlap between hnRNP H1 and F (2,821 mRNAs; Fig. 4B), which is not unexpected based on previous studies of hnRNP H1 and F interacting mRNAs (40,41).…”

“…4E). A/G-rich motifs were also identified as direct binding motifs for hnRNP H1 in HEK 293T and HeLa cells (40,41). Binding to Hmgb1 mRNA was only identified HEK 293T cells, and that was an intronic RNA interaction rather than to the mRNA (44); Nrn1 mRNA was not found in either cell line possibly due to low Nrn1 expression (40,41).…”

RNA-binding proteins in neurodegeneration: mechanisms in aggregate

“…Importantly, this correlated with missplicing of many known hnRNP H target exons (Conlon et al 2016). hnRNP H levels affect splicing of hundreds of targets , including many encoding proteins involved in splicing-among them TDP-43 and FUS (Uren et al 2016). Thus, hnRNP H deficiency may result in splicing errors that extend beyond exons with which hnRNP H interacts physically.…”

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC

“…Here, we delineate a refined set of mRNA targets by integrating different genomic approaches. A similar strategy significantly reduced the mRNA target space for HuR and hnRNP H1(Uren et al, 2016, Mukherjee et al, 2011). This integrated approach to elucidating IGF2BP3 target transcripts was also applied to human B-ALL cells (Palanichamy et al 2016).…”