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Genetic Control of pre-mRNA Splicing and Diseases

Zhi-xiang Lu

Department of Microbiology, Immunology, and Molecular Genetics, University of California

Corresponding author

Zhi-xiang Lu, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA, E-mail: zhixianglu@ucla.edu

Received Date: 22 May 2014

Accepted Date: 25 May 2014

Published Date: 28 May 2014


Lu ZX (2014) Genetic Control of pre-mRNA Splicing and Diseases Enliven: J Genet Mol Cell Biol 1(1): e3.


Dr. Zhi-xiang Lu. This is an Open Access article published and distributed under the terms of the Creative Commons Attribution License, that permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

What Is Alternative Splicing?

Eukaryotic genes, primarily in multi-cellular organisms, have mRNA-encoding regions called exons that are interrupted by non-coding regions named introns. This split gene structure provides a fertile ground of post-transcriptional regulation which expands transcript repertoire through usage of various exon-exon combinations (i.e. alternative splicing) resulting in multiple mRNA isoforms (i.e. splice variants) produced from a single gene [1]. High-throughput RNA sequencing (RNA-seq) indicates that > 90% of multi-exon human genes undergo alternative splicing (AS) [2,3]. More importantly, AS can change a final protein’s binding properties, modify enzymatic activity, and even reverse roles of its gene in cellular processes (e.g. two isoforms of Bcl-x through AS, the long form is anti-apoptotic, while the short one is pro-apoptotic) [4]. Besides the role of AS in normal development, evidence of pathogenesis and clinical relevance of aberrant splicing variants is growing exponentially [5], with an estimated 10% of splice site mutations (exon intron boundary) causing human inherited diseases [6-8]. Deciphering the pre-mRNA splicing code and functional characterization of splice variants will provide us new insights of pathogenesis of human diseases.

How Is pre-mRNA Splicing Regulated?

The precision of pre-mRNA splicing relies on interactions between cis elements and trans regulators that recognize cis elements (Figure 1). The core cis splicing signals include two groups of elements: (1) the 5’ or 3’ splice site which determines the exon-intron boundary; (2) branch site and polypyrimidine tract that are initially recognized during intron cleavage steps. Other auxiliary exonic and intronic elements (i.e. ESE: exonic splicing enhancer, ISE: intronic splicing enhancer, ESS: exonic splicing silencer, ISS: intronic splicing silencer) also participate in the process, either promoting orinhibiting splicing. During the splicing process, the core splicing signals (e.g. 5’ or 3’ splice site) are recognized by spliceosome complex which is composed of five small nuclear RNAs (snRNAs: U1, U2, U4, U5, and U6) and associated protein factors, while auxiliary element region (e.g. ESE or ISS) can recruit SR (serine/arinine-rich)-proteins and hnRNPs (heterogeneous nuclear ribonucleoproteins). Interplay of RNA-protein and protein-protein interaction leads to final precise splicing in normal development [9].

Pre-mRNA Splicing Defects in Diseases

Either mutation of cis elements or perturbation of trans factors could impair splicing which contributes to disease phenotypes [7]. For instance, the occurrence of congenital bilateral absence of vas deferens (CBAVD) and full brown cystic fibrosis disease is associated by loss of CFTR (Cystic fibrosis transmembrane conductance regulator) exon 9, an aberrant alternative splicing with production of an inactive CFTR protein. Further studies proved that the TG(m)T(n) polymorphic element in the vicinity of CFTR exon 9 affects the efficiency of its exon 9 splicing [10]. Another example of cis element mutation affecting splicing is in ataxia telangiectasia, a neurodegenerative disease. Baralle and his colleagues found that deletion of four nucleotides (GTAA) in intron 20 of ATM gene abolished accurate intron processing, caused activation of the cryptic exon of 65 bp [11]. The ATM allele with this 4nt deletion fails to produce a functional protein, which is vital for cell cycle checkpoint signalling in DNA damage response and genome instability. Among the diseases caused by splicing trans regulators, myotonic dystrophy (DM1) is a particularly well-studied example. An expanded CUG repeat in 3’ UTR of DMPK gene leads to sequestration of MBNL protein and upregulation of the fetal splicing patterns controlled by CELF protein, which finally contributes to severe manifestations of disease [12]. Interestingly, disease severity can by modulated by the interplay of cis mutation and trans factor perturbation. One splicing genetic modifier of SCN8A gene has been characterized in mice. In C3H mice, a 4nt deletion (GTAA) close to 5’ splice site of SCN8A exon 3 results in skipping of both exon 2 and exon 3 in most transcripts resulting in only 10% of the transcripts beingcorrectly spliced. C57BL/6J mice, in addition to the 4nt deletion in SCN8A gene, have a stop mutation in SCNM1 gene whose protein product acts as a splicing factor controlling SCN8A splicing patterns. With both the deletion in SCN8A and the mutation in SCNM1, only 5% of SCN8A will be correctly spliced in C57BL/6J mice. This small (10% vs. 5%) splicing difference however leads to dramatically distinct disease phenotypes. C3H mice only have a disorder of chronic movement while C57BL/6J mice develop a severe lethal neurological disease [13]. Such above evidence underscores the roles of AS in inherited diseases. In addition, aberrant splicing events observed in cancers through alteration of splicing factor concentration or localization are reviewed extensively elsewhere [14,15].

Perspectives and Challenges of Studying Defective Splicing

Despite widespread use of AS in both major physiological and pathological aspects of cell biology, we still do not understand the function of most splice variants. Without functional data, we cannot decipher if these aberrant splicing events play a role in initiation and/or development of diseases or whether they are just “passenger” isoforms. In fact, functional characterization of certain transcript on a splicing level involves more technical difficulties than the gene level. For instance, confounding factors cannot be ruled out, such as global gene expression change, or additional isoform product from cryptic exon usage when the target exon splicing of interest is blocked. Moreover, due to the function redundancy of splicing regulatory proteins it is extremely difficult to get clear regulatory architecture of certain defective splicing. Thanks to cutting-edge technologies such as deep RNA sequencing and clinical whole exome sequencing, we are now able to access the potential links between AS, DNA variants and disease traits [16]. Furthermore, many splicing regulatory elements that promote or inhibit splicing have been identified in in vitro random oligonucleotide library screening [17,18]. Currently, HITS-CLIP (high throughput sequencing by crosslinking immunoprecipitation) provides us a refined interaction map between mRNA and RNA binding proteins across diverse tissues and cell types [19,20]. All above comprehensive studies are facilitating many important investigations of AS regulation. For example, globally correlating AS to RNA binding map of certain splicing factor scan gain important insights into how RNA-protein interactions affect splicing, integration of hundreds of putative splicing regulatory elements/feature can predict tissue-specific AS patterns [21,22]. Meanwhile, antisense oligonucleotide targeting splice sites have been developed as a modulator of pre-mRNA splicing, a powerful approach to dissect function role of splicing variants [23]. Some promising antisense oligonucleotides are already in clinical trials, for instance AVI-4658 is in clinical trials testing for mis-splicing disorder in Duchenne Muscular Dystrophy [24]. We believe that this RNA splicing topic will deepen the impression of AS roles in diseases and foresee the AS biomarkers as the effective therapeutic targets.


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