Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5' splice site selection.
Bottom Line: Binding of PTBP1 to this site was required for its effect on splicing.Notably, a similar function of PTBP1 in the selection of alternative 5' splice sites was confirmed using the USP5 gene as additional model.Our study provides a novel mechanism of alternative 5' splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5' splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator.
Affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy Laboratory of Neuroembryology, Fondazione Santa Lucia, 00143 Rome, Italy.Show MeSH
Related in: MedlinePlus
Mentions: We found that concomitant knockdown of PTBP1/2 and hnRNP F did not exert additive effects (Supplementary Figure S4C). Moreover, co-overexpression of PTBP1 and hnRNP F elicited only a mild additive effect (Supplementary Figure S4D), indicating that the two splicing factors do not cooperate in the regulation of BCL-X AS. Next, we asked whether PTBP1 functions by antagonizing SRSF1 activity. First, we performed CLIP assays in HEK293T to evaluate whether SRSF1 directly binds BCL-X RNA in vivo. SRSF1 was enriched near the proximal 5′ splice site (E2p) (Figure 5A), indicating that it binds the BCL-X RNA just downstream of PTBP1 in vivo. Moreover, splicing assays using the wt minigene and suboptimal amounts of SRSF1 showed that increasing PTBP1 expression efficiently antagonizes SRSF1 activity in a dose-dependent manner (Figure 5B). By contrast, competition was lost when splicing assays were performed using the E2m1 minigene, in which the PTBP1 binding site is disrupted (Figure 5B). Likewise, in the presence of suboptimal amounts of PTBP1, SRSF1 antagonizes its activity in a dose-dependent manner (Supplementary Figure S5A). To test whether this competition was the result of a direct effect, we performed CLIP assays. In HEK293T cells transfected with the wt BCL-X minigene, PTBP1 was recruited efficiently to the BCL-X RNA whereas SRSF1 was barely detectable (Figure 5C). However, when cells were transfected with the E2m1 minigene, PTBP1 binding was significantly reduced whereas SRSF1 was recruited more efficiently (Figure 5C).
Affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy Laboratory of Neuroembryology, Fondazione Santa Lucia, 00143 Rome, Italy.