Regulation of alternative splicing by PTB and associated factors

Abstract

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

Caffeine Regulates Alternative Splicing in a Subset of Cancer-Associated Genes: a Role for SC35

“…3A, upper panel); this resulted in greater than 90% depletion of hUpf1 protein by 72 h posttransfection. A splice variant of the polypyrimidine transcript binding protein (PTB), known to be a target of NMD (48), was used as a positive control for NMD inhibition (Fig. 3A, left panel).…”

“…After 18 h, 25 to 100 nM small interfering RNA (siRNA; siGENOME SMARTpool reagent; Dharmacon) was mixed with Oligofectamine reagent (Invitrogen), incubated at room temperature for 20 min, and added drop-wise to cells. At 48,72, and 96 h post-siRNA transfection, cells were harvested for either whole-cell lysates or RNA isolation. The concentration and time required for optimal downregulation were determined empirically by both RT-PCR and Western blot analysis.…”

The Coupling of Alternative Splicing and Nonsense-Mediated mRNA Decay

“…67 Consequently, when PTB levels are high, PTB production is slowed by targeting PTB transcripts for NMD and when PTB levels are low, production is accelerated by reducing the proportion of transcripts that are degraded. 67,69,70 A similar autoregulatory process has been reported for members of a family of splicing factors known as SR proteins. Overexpression of the SR protein SC35 upregulates the splicing of its own NMD-targeted isoform to reduce protein production.…”

SR protein 9G8 modulates splicing of tau exon 10 via its proximal downstream intron, a clustering region for frontotemporal dementia mutations

“…Instead, we discovered that 9G8 exerts its effect by binding to the intronic splicing silencer defined by deletion Δ11/18 and by a bevy of FTDP mutations (M11, 12, 13, 14, 16). This is unusual: in the traditional division of labor, SR proteins act on exonic elements whereas hnRNP and CELF proteins act on intronic elements (Sanford et al, 2005;Spellman et al, 2005). However, SR proteins also act as bridging elements for regulated exons (Sanford et al, 2005) and 9G8 may be fulfilling that role in this instance.…”