Growth Hormone Deficiency and Splicing Fidelity

Abstract

The majority of mutations that cause isolated growth hormone deficiency type II are the result of aberrant splicing of transcripts encoding human growth hormone. Such mutations increase skipping of exon 3 and encode a 17.5-kDa protein that acts as a dominant negative to block secretion of full-length protein produced from unaffected alleles. Previously, we identified a splicing regulatory element in exon 3 (exonic splicing enhancer 2 (ESE2)), but we had not determined the molecular mechanism by which this element prevents exon skipping. Here, we show that two members of the serine/arginine-rich (SR) protein superfamily (ASF/SF2 and SC35) act antagonistically to regulate exon 3 splicing. ASF/SF2 activates exon 3 inclusion, but SC35, acting through a region just downstream of ESE2, can block such activation. These findings explain the disease-causing mechanism of a patient mutation in ESE2 that creates a functional SC35-binding site that then acts synergistically with the downstream SC35 site to produce pathological levels of exon 3 skipping. Although the precedent for SR proteins acting as repressors is established, this is the first example of a patient mutation that creates a site through which an SR protein represses splicing.Pre-mRNA splicing is the process of intron removal and exon joining to form mature, protein-coding transcripts. This requires both cis-acting RNA elements and trans-acting factors. The main cis-acting elements are the 5Ј splice site, 3Ј splice site, polypyrimidine tract, and branch point sequence, each of which are defined by short, degenerate consensus sequences in higher eukaryotes (1, 2). Recognition of these elements by trans-acting factors, including five small nuclear ribonucleoproteins, results in assembly of the catalytically active spliceosome in which the two steps of splicing occur. Because of the lack of sequence conservation, the pairing of U1 snRNA with the 5Ј splice site and U2 with the branch point results in a range of splice site strengths. This allows for regulation of splice site selection in cases of alternative splicing but at the same time creates a potential fidelity problem in cases where weak splice sites need to be properly recognized. These weak splice sites often require additional sequences known as enhancer elements that are recognized by regulatory splicing factors, such as SR 3 proteins, which guide the spliceosome to the correct splice sites. Splicing enhancers are typically purinerich and can be found in both exons and introns and are accordingly named exonic (ESEs) and intronic splicing enhancers. SR proteins are a large family of conserved proteins that have N-terminal RNA recognition motifs and C-terminal domains rich in serine-arginine dipeptides (RS domains). The RNA recognition motifs tether the proteins to enhancer elements, and the RS domains assist spliceosome assembly through proteinprotein interactions (3).SR proteins define exon boundaries by two mechanisms that are not mutually exclusive. First, SR proteins enhance splicing in an RS domain-i...

Regulation of splicing by SR proteins and SR protein-specific kinases

“…Because alternative splicing is the choice of splice sites in competition, it has also been demonstrated (depicted in Figure 1B, panel b) that SR protein binding to the alternative exon promotes exon inclusion whereas SR protein binding to a flanking exon causes skipping of the internal alternative exon (Han et al, 2011a; Sanford et al, 2009). This pair of rules may explain antagonizing activities of different SR proteins in some specific alternative splicing events (Gallego et al, 1997; Ghigna et al, 2005; Lemaire et al, 1999; Solis et al, 2008), which is consistent with recent genome-wide analyses (Pandit et al, 2013; Sanford et al, 2009). …”

Regulation of alternative splicing within the supraspliceosome

“…The mechanism underlying this effect is not yet well understood. In some cases, however, it was attributed to intronic binding sites for the SR proteins, or to the ESEs in other cases (Buratti et al, 2007; Gallego et al, 1997; Jiang et al, 1998; Lemaire et al, 1999; Solis et al, 2008; ten Dam et al, 2000). In a recent study it was shown that the exon skipping effect of SR proteins depends on their actions on a flanking constitutive exon and also requires joint effort of more than one SR protein (Han et al, 2011).…”

Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

“…Introns bins correspond to (1) <350nt, (2) 350nt-1kb, (Solis et al, 2008) 1kb-2kb, (4) 2kb-4kb, and (5) >4kb.…”

“…Through binding to ESEs, SR proteins function as positive splicing regulators to promote exon inclusion. However, increasing evidence suggests that they are also involved in exon-skipping events (Gallego et al, 1997; Ghigna et al, 2005; Lemaire et al, 1999; Solis et al, 2008), possibly through competition between the alternative exon and flanking constitutive exons (Sanford et al, 2009). For example, tethering an SR protein to the alternative exon induces its inclusion, but anchoring the SR protein in a flanking exon promotes alternative exon skipping (Han et al, 2011a).…”