RNA structure in alternative splicing regulation: from mechanism to therapy

Abstract

Alternative splicing is a highly intricate process that plays a crucial role in post-transcriptional regulation and significantly expands the functional proteome of a limited number of coding genes in eukaryotes. Its regulation is multifactorial, with RNA structure exerting a significant impact. Aberrant RNA conformations lead to dysregulation of splicing patterns, which directly affects the manifestation of disease symptoms. In this review, the molecular mechanisms of RNA secondary structure-mediated splicing regulation are summarized, with a focus on the complex interplay between aberrant RNA conformations and disease phenotypes resulted from splicing defects. This study also explores additional factors that reshape structural conformations, enriching our understanding of the mechanistic network underlying structure-mediated splicing regulation. In addition, an emphasis has been placed on the clinical role of targeting aberrant splicing corrections in human diseases. The principal mechanisms of action behind this phenomenon are described, followed by a discussion of prospective development strategies and pertinent challenges.

1. Introduction

The remarkable biological complexity of eukaryotes is largely attributable to the intricate regulatory networks that dynamically control protein isoform expression in a spatio-temporal manner. As an integral part of gene expression, alternative splicing is a form of post-transcriptional regulation capable of generating diverse mature RNA transcripts and functional protein isoforms from a single gene. Alternative splicing was first reported for the mRNA of adenovirus 2 and was soon proven to be a prevalent mechanism across eukaryotes [1]. Moreover, organismal complexity showed a strong positive correlation with the proportion of genes undergoing alternative splicing [2]. Species with greater tissue and cellular heterogeneity tend to exhibit higher levels of alternative splicing events on a genome-wide scale. Notably, approximately 95% of human genes are estimated to undergo alternative splicing. Furthermore, since a substantial proportion of alternative splicing events are tissue specific, the resulting splicing isoforms often exhibit subtle or radical differences in functional attributes [3]. Consequently, alternative splicing plays an indispensable role in cellular differentiation and organismal development by increasing the functional diversity of the transcriptome and proteome.

The regulation of pre-mRNA splicing is a complex multistep process. This process entails precise excision of introns and the joining of exons, both of which are orchestrated by a highly complex macromolecular assembly known as the spliceosome. Previous extensive reviews have elucidated the mechanisms underlying spliceosome assembly and pre-mRNA splicing [4‒6]. In brief, the spliceosome recognizes and binds to the exon-intron junctions (5′-splice site and 3′-splice site) of nascent pre-mRNAs, as well as to intronic branch points and polypyrimidine tracts. This facilitates exon recognition and executes two successive transesterification reactions, resulting in intron removal and exon ligation. The core of the process is the definition of the 5′- and 3′-splice sites of the exon. Based on differences in exon definitions, alternative splicing events can be categorized into the following five types: exon skipping, intron retention, alternative 5′ or 3′ splice site usage, and mutually exclusive splicing [1]. Remarkably, human introns range in length from several to hundreds of kilobases, with an average size of ~5 kb, and they are interspersed with numerous ‘decoy’ splice sites that show significant consensus sequence similarity with authentic splice sites. These ‘decoy’ splice sites have the potential to induce the inclusion of pseudo-exons [7,8]. Despite the prevalence of decoys, splicing proceeds with extraordinary fidelity, and it is strongly argued that additional sequence features surrounding core splicing signals contribute to exon-intron definition [9].