Decoding HNRNPH-mediateed splicing regulation

Abstract

Alternative splicing is one of the most important molecular pathways for a cell to create multiple protein-coding mRNA isoforms from one gene, contributing to the proteomic complexity and enabling cellular diversity. An interplay of RNA-binding proteins (RBPs) guides alternative splicing decisions in human cells. Heterogeneous nuclear ribonucleoprotein H (HNRNPH) is one of these RBPs, which functions as a splice factor. Recently, Braun et al. (2018) showed that HNRNPH cooperatively regulates alternative splicing of the proto-oncogene MST1R. Such a cooperative splicing regulation would make the cellular system very sensitive to even only small changes in the HNRNPH expression. Inspired by these results, the here presented project aimed to analyse a transcriptome-wide HNRNPH-mediated cooperative splicing regulation and to decode the underlying mechanism. Initial RNA-Sequencings (RNA-Seq) from cells with titrated HNRNPH protein levels highlighted indeed that HNRNPH regulates alternative splicing cooperatively on a transcriptome-wide scale. To decode the cooperative mechanism, an Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiment showed that HNRNPH binds to RNAs composed of guanine (G)-rich sequences overlapping with predicted RNA G-quadruplexes (rG4). In vitro Reverse Transcriptase Stop Profiling validated rG4 structure formation of HNRNPH binding sites in high-throughput. Next, in vitro iCLIP revealed that HNRNPH has an intrinsic preference for the binding to RNAs with rG4-folding potential and in vitro binding studies showed that rG4 structure formation is required for cooperative binding of HNRNPH. Finally, in vivo minigene studies highlighted the involvement of rG4s in cooperative splicing regulation by HNRNPH in cells. In summary, this study describes for the first time that HNRNPH mediates alternative splicing cooperatively on a transcriptome-wide scale and proposes the importance of rG4 structures in this cooperative regulation.