An intronic element contributes to splicing repression in spinal muscular atrophy

Abstract

The neurodegenerative disease spinal muscular atrophy is caused by mutation of the survival motor neuron 1 (SMN1) gene. SMN2 is a nearly identical copy of SMN1 that is unable to prevent disease, because most SMN2 transcripts lack exon 7 and thus produce a nonfunctional protein. A key cause of inefficient SMN2 exon 7 splicing is a single nucleotide difference between SMN1 and SMN2 within exon 7. We previously provided evidence that this base change suppresses exon 7 splicing by creating an inhibitory element, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer. We now find that another rare nucleotide difference between SMN1 and SMN2, in intron 7, potentially creates a second SMN2-specific hnRNP A1 binding site. Remarkably, this single base change does indeed create a highaffinity hnRNP A1 binding site, and base substitutions that disrupt it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding in vitro. We propose that interactions between hnRNP A1 molecules bound to the exonic and intronic sites cooperate to exclude exon 7 and discuss the significance of this exclusion with respect to SMN expression and splicing control more generally.alternative splicing ͉ exonic splicing silencer ͉ hnRNP A1 ͉ intronic splicing silencer A lternative splicing of mRNA precursors is an important mechanism that regulates gene expression and generates increased protein diversity from a limited number of genes. Indeed, expression of 70% or more of human genes is now estimated to involve alternative splicing (1, 2). In higher eukaryotes, alternative splicing plays an essential role in diverse cellular functions, contributing to such basic processes as cell growth, differentiation, and cell death (3, 4). Several types of cis-acting RNA elements and trans-acting protein factors help to regulate alternative splicing and do so by diverse mechanisms. In general, however, non-spliceosomal proteins that participate in regulating alternative splicing associate with regulatory elements in exons and/or introns. Serine/arginine-rich proteins (5) are typically involved in positive regulation of splicing, stimulating splicing by interacting with exonic splicing enhancer elements (ESEs) or intronic splicing enhancer elements. In contrast, negative regulation is promoted most frequently by heterogeneous nuclear ribonucleoproteins (hnRNPs) (6), which function by binding sequences known as exonic splicing silencers (ESSs) or intronic splicing silencers.Several hnRNP proteins have been identified as key splicing repressors. Among these, the abundant hnRNP A1 protein has been extensively characterized. The first hnRNP A1-dependent ESS was identified in studies of HIV tat exon 2 repression (7-9). This ESS was found to bind hnRNP A1, and mutations disrupting the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing. Several mechanisms have been proposed to explain hnRNP A1-mediated splicing repression. In one, hnRNP A1 binds to an ESS and through direct protein-protein interactions, recruits more ...

Alternative splicing in spinal muscular atrophy underscores the role of an intron definition model

“…For example, a SMN2 specific A-to-G mutation at the 100 th position (A100G) of intron 7 creates an hnRNP A1 motif. 58 Of note, A100G is a deep intronic mutation that does not create a cryptic splice site and yet imparts a significant impact on SMN2 exon 7 splicing. It is likely that hnRNP A1 bound to this motif (at the 100 th position of intron 7) communicates with another hnRNP A1 molecule interacting with ISS-N1 and/or C6U location.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…The regulation of pre-mRNA splicing was one of the earliest known functions of hnRNP A1, a function it often carries out by binding to ESS sequences and repressing exon inclusion (Mayeda and Krainer 1992;Del GattoKonczak et al 1999). hnRNP A1 bound to ISS sequences can also promote exon exclusion, possibly through a mechanism that involves self-interaction of A1 molecules bound to distal sites and loop formation (Blanchette and Chabot 1999;Kashima et al 2007). While best characterized as repressors of splicing, hnRNP A/B proteins can also activate inclusion of some exons (Martinez-Contreras et al 2006;Venables et al 2008).…”

Differential 3′ splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP

“…Exon 7 skipping generates a truncated, SMN2D7 protein isoform, which can only provide limited SMN function (Le et al 2005). Several nucleotide differences exist between the genomic sequence of SMN1 and SMN2 genes, and two of them have been implicated in increased skipping of exon 7 in SMN2 transcripts, a critical translationally silent C to T transition at position 6 of exon 7 and an A to G transition at position 100 of intron 7 (Kashima et al 2007a). Because the extent of SMN2 exon 7 skipping, which is variable in SMA patients, has been correlated with disease severity (Mailman et al 2002), modulation of this splicing event has been proposed as a potential therapy for the disease.…”