Dynamic Regulation of Alternative Splicing by Silencers that Modulate 5′ Splice Site Competition

Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
Cell (Impact Factor: 32.24). 01/2009; 135(7):1224-36. DOI: 10.1016/j.cell.2008.10.046
Source: PubMed


Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with approximately 70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5' splice site located downstream of a weakened 5' splice site. We recovered two exonic and four intronic motifs that effectively silenced the proximal 5' splice site both in vitro and in vivo. Surprisingly, silencing was only observed in the presence of the competing upstream 5' splice site. Biochemical evidence strongly suggests that the silencing motifs function by altering the U1 snRNP/5' splice site complex in a manner that impairs commitment to specific splice site pairing. The data indicate that perturbations of non-rate-limiting step(s) in splicing can lead to dramatic shifts in splice site choice.

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Available from: Xiang Zhang, Mar 04, 2014
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    • "Alternative splicing is modulated by combinatorial control exerted by overlapping linear motifs called exonic or intronic splicing enhancers and silencers (ISSs) (1–3). Although methods to define linear splicing motifs continue to evolve (4–6), there is a growing appreciation of the role of RNA structure in regulation of alternative splicing (7–10). RNA secondary structure folding occurs on a microsecond time scale (11,12), a rate that is faster than polymerase II-mediated transcription elongation, which is ∼100 nt per second (13). "
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    ABSTRACT: Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2′-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3′ strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3′ strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.
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    • "Exon encodes part of the ligand binding domain and its skipping abolishes receptor activity (Chen et al., 2005b) NCOR2 (nuclear receptor corepressor 2) Alternative splicing generates two isoforms with different affinities for nuclear receptors (Goodson et al., 2005) carm1 (coactivator-associated arginine methyltransferase 1) Xenopus laevis Alternative usage exon 14 generates isoforms with opposite effects on ligand-mediated transcription (Matsuda et al., 2007) THRB (thyroid hormone receptor, beta) Stop codon in ligand binding domain creates inactive receptor/transcription factor (Tagami et al., 2010) Nr1i2 (nuclear receptor subfamily 1, group I, member 2) Mus musculus Loss of 41 aa adjacent to ligand binding pocket represses function of full-length form (Matic et al., 2010) E. Change in intracellular localization ESRRB (estrogen-related receptor beta) Change in F-domain that alters nuclear localization and ligand binding (Zhou et al., 2006) Gtf2i (general transcription factor II-I) Mus musculus On/off switch after change in localization due to cell stimulation (Hakre et al., 2006) NOSTRIN (nitric oxide synthase trafficker) Truncated isoform has nuclear rather then cytoplasmic localization and negatively regulates transcription of its own gene (Wiesenthal et al., 2009) ESRRB (estrogen-related receptor beta) Alternative sequence at the C-terminus influence intra-nuclear mobility and activation of reporter genes (Bombail et al., 2010) (continued on next page) non-coding RNA in exon selection. These RNA elements can be located on the pre-mRNA and regulate the binding of U1 snRNP by stabilizing the interaction of U1 with competing 5′ splice sites (Yu et al., 2008). The RNAs can also be generated from other transcripts and then regulate pre-mRNAs (reviewed in Khanna and Stamm, 2010). "
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    ABSTRACT: Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.
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    • "Such auxiliary sequences, which stimulate splicing are found in both exons (exonic splicing enhancers, ESE), and introns (intronic splicing enhancers, ISE; Aznarez et al., 2008; Lomelin et al., 2010; Brooks et al., 2011). Moreover, sequences that inhibit splicing have also been characterized, as exonic and intronic splicing silencers (ESS and ISS; Yu et al., 2008; Zhang et al., 2008; Wen et al., 2010). Additionally, the sequence complexity of these splicing regulatory elements is highlighted by the in vivo analysis of all possible hexamers as exonic splicing regulators, which revealed that out of the 4,096 combinations more than half could act as either ESE or ESS (Ke et al., 2011). "
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    ABSTRACT: Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma-associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation.
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