Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins

Abstract

SUMMARY Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

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

“…15 Genome-wide profiles have favored the former, correlating longer poly-G stretches with more confident changes in splicing, 6 and interspersion or termination of the binding site by adenosine. 2 Our data, however, is supportive of the TGGG tetramer as a prevalent component of the hnRNP H1 binding motif, with particular enrichment in intronic regions. When we looked for occurrences of these motifs around the iCLIP cross-link locations however, we noticed that enrichment was quite diffuse (data not shown), with no strong enrichment right at the iCLIP site.…”

“…8 Other recent studies have linked hnRNP H1 to alternative cleavage and polyadenylation, 6 and established the cooperative roles of the hnRNP proteins in the regulation of splicing. 2 By employing a combined strategy with 4 different high-throughput approaches, we were able to provide a more refined and accurate picture for hnRNP H1 in splicing regulation and extend our understanding of its participation in polyadenylation and mRNA decay. Moreover, our examination of the hnRNP H1 target set has uncovered its role as a key regulator of RNA processing genes and identified novel routes by which it may contribute to cancer development and diseases.…”

“…Previous results that suggested an even broader involvement of hnRNP H1 in direct splicing should be taken with some degree of caution as both less stringent approaches to target calling, and large window distances for correlating binding sites and changes in splicing were used. 2 Fig. 4A shows the fold-change and significance of changes in exons with proximal hnRNP H1 sites and the ones that do not.…”

“…This protein tends to form a heterodimer with hnRNP F and shows preference for G-rich tracts of RNA. 2,[6][7][8] It has also been shown that hnRNP H1 interacts with the DGCR8 complex and facilitates microRNA processing in HeLa cells. 9 The impact of hnRNP H1 binding on alternative splicing has been associated with several diseases, such as cystic fibrosis, 10 congenital myasthemic syndrome, 11 and amyotrophic lateral sclerosis (ALS).…”

“…Although the most well-known function of the family is direct involvement in alternative splicing events, 2-4 they also participate in telomere maintenance, transcription, polyadenylation, mRNA trafficking, mRNA degradation, and translational regulation. 2 Domain composition among hnRNP members is highly diverse. Some show classical RRM or KH domains, 3 while hnRNP H1, hnRNP H2 and hnRNP F exhibit the so called quasi-RRMs (or qRRM) domains.…”