MBNL splicing activity depends on RNA binding site structural context

Abstract

Muscleblind-like (MBNL) proteins are conserved RNA-binding factors involved in alternative splicing (AS) regulation during development. While AS is controlled by distribution of MBNL paralogs and isoforms, the affinity of these proteins for specific RNA-binding regions and their location within transcripts, it is currently unclear how RNA structure impacts MBNL-mediated AS regulation. Here, we defined the RNA structural determinants affecting MBNL-dependent AS activity using both cellular and biochemical assays. While enhanced inclusion of MBNL-regulated alternative exons is controlled by the arrangement and number of MBNL binding sites within unstructured RNA, when these sites are embedded in a RNA hairpin MBNL binds preferentially to one side of stem region. Surprisingly, binding of MBNL proteins to RNA targets did not entirely correlate with AS efficiency. Moreover, comparison of MBNL proteins revealed structure-dependent competitive behavior between the paralogs. Our results showed that the structure of targeted RNAs is a prevalent component of the mechanism of alternative splicing regulation by MBNLs.

Expanded CUG repeats in DMPK transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences

“…The branched hairpin model has implications for understanding aberrant protein interaction with expanded repeats, a central theme in the study of DM1 etiology. MBNL1 preferentially binds its epitope YGCY in an unstructured environment, which is abundant in the branched, dynamic structures that we propose, but rare in perfect hairpins (Taylor et al 2018). This notion could answer the question raised before on how MBNL1 normally interacts with mostly single-stranded targets, but nonetheless binds CUG repeat RNA that was considered highly structured (Cass et al 2011).…”

RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

“…The RNA structure context was discussed as being able to affect protein binding [70]. Motif analysis of the retained introns of nucleus-localized transcripts detetced 14 enriched RBPs, in which MBNL1 has been reported to bind RNA in the CUG repeats [71] and contain two nuclear signals [72]. Previous studies have reported that the CUG repeat expansion caused nuclear aggregates and RNA toxicity, which in turn induces neurological diseases [73,74].…”

Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control

“…A double-stranded RNA-binding protein Staufen homolog 1 (STAU1) was found to stabilize RNA structures upon binding after RNA leaves chromatin, whereas hnRNP C binding to RNA was correlated with the structural disturbance of flanking regions [ 50 ]. The aforementioned AS factor, MBNL1, forms functional complexes with specific linear and structural RNA motifs [ 172 ] but upon binding it most likely unwinds the local secondary structure of the RNA as it was shown in an in vitro footprinting assay [ 173 ].…”