The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches

Abstract

Removal of introns from pre-mRNA precursors (pre-mRNA splicing) is a necessary step for the expression of most genes in multicellular organisms, and alternative patterns of intron removal diversify and regulate the output of genomic information. Mutation or natural variation in pre-mRNA sequences, as well as in spliceosomal components and regulatory factors, has been implicated in the etiology and progression of numerous pathologies. These range from monogenic to multifactorial genetic diseases, including metabolic syndromes, muscular dystrophies, neurodegenerative and cardiovascular diseases, and cancer. Understanding the molecular mechanisms associated with splicing-related pathologies can provide key insights into the normal function and physiological context of the complex splicing machinery and establish sound basis for novel therapeutic approaches.

Keywords: RNA; alternative splicing; disease; mutation; spliceosome.

Figure 1. Mechanisms of splice site recognition and exon definition

(A) Splicing complex assembly is initiated by consensus sequence elements located at the exon (blue)/intron (brown) boundaries. Recognition of the 5′ SS by U1 snRNP involves base‐pairing interactions between the 5′ end of U1 snRNA. Recognition of the 3′ SS region involves binding of the U2AF65/35 heterodimer to the polypyrimidine tract (Poly‐Y tract) and conserved 3′ SS, which facilitates recruitment of U2 snRNP to the branch site, involving base‐pairing interactions between U2 snRNA and nucleotides flanking the branch point adenosine. (B) Exon definition modulated by exonic and intronic sequence elements, which can promote (ESE & ISE, in orange) or suppress (ESS & ISS, in dark red) splice site recognition. A classic model involves recognition of splicing enhancers by proteins of the SR family and recognition of splicing silencers by hnRNP proteins. However, proteins of these and other families can promote or inhibit splicing depending upon the location of their binding sites relative to the splice sites and other regulatory sequences. A complex combinatorial interplay between regulatory elements and their cognate factors determines exon definition and regulation. (C) A switch from stabilizing interactions between factors recognizing 3′ and 5′ SS across exons (exon definition) to splice site pairing (intron definition) and tri‐snRNP assembly occurs in vertebrate internal exons and can be targeted by regulatory factors like hnRNP L or RBM5.

Figure 2. The spliceosome assembly pathway

Initial recognition of the 5′ SS by U1 snRNP and of the 3′ SS region by SF1 (branch point binding protein) and U2AF (complex E) is followed by the ATP‐dependent recruitment of U2 snRNP to the branch point region (complex A), concomitant with displacement of SF1. Binding of the U4/5/6 tri‐snRNP and the protein‐only NineTeen Complex (NTC) leads to formation of complex B, involving also the displacement of proteins present in complex A. Extensive remodeling of complex B, including the destabilization/displacement of U1 and U4 snRNPs, leads to a catalytically active complex (Bact), which upon further conformational rearrangements and changes in protein composition catalyzes the first step of the splicing reaction, leading to the formation of a lariat intermediate containing a 2′‐5′ phosphodiester bond (complex C). An additional conformational switch leads to the second catalytic step, rendering the spliced product and the intron lariat. Upon release of the products, the lariat intron is linearized and degraded and the snRNPs recycled for another round of assembly and catalysis in other introns. Proteins of the ATP‐dependent DEAH/X helicase family (in magenta) are key to promote the multiple conformational transitions, characterized by extensive rearrangements of RNA:RNA interactions involving snRNA:snRNA and snRNA:pre‐mRNA contacts.

Figure 3. Spectrum of pathologies associated with splicing defects

Broadly classified as mutations in sequence elements (A), alterations in splicing factors (B) and titration/signaling effects of nucleotide repeat expansions (C), the nature of various molecular alterations and their effects on splicing are described for various pathologies associated with splicing defects.

Figure 4. Summary of therapeutic approaches based upon splicing modulation

Two main strategies are outlined. Splice‐switching antisense oligonucleotides target splice sites or splicing regulatory sequences to prevent the binding of cognate factors to modulate splice site selection. Examples include blocking of an ISS element that promotes exon 7 inclusion in the SMN2 gene, leading to restoring SMN protein expression and motoneuron function in SMA; induction of skipping of a mutation‐containing exon in the DMD gene, leading to in‐frame deletion of a nonessential part of the dystrophin protein and restoration of muscle function; prevention of sequestration of the MBNL splicing regulator in CUG repeat expansions in DMPK transcripts, leading to restoration of abnormal splicing patterns in DM. Small molecules, some of them targeting core splicing components like SF3B, modulate alternative splicing of cell cycle control genes and display anti‐tumoral properties. Other drugs can induce SMN2 exon 7 inclusion and therefore raise hope as oral treatments for SMA.