Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

Abstract

Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5' splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection.

FIGURE 1.

The positioning of splicing regulatory elements determines their effect on 5′ splice site (5′ss) activation. (A) Schematic drawing of the RNA splicing reporter. Specific binding sites for splicing regulatory proteins are immediately upstream of or downstream from the D1 5′ss, as indicated by boxes. HeLa cells were transiently transfected with each of the reporter constructs and analyzed by semi-quantitative and real-time PCR to determine the position-dependent activity of SREs (B) and to demonstrate that each SRE displays position-dependent activation and repression of splicing (C). N1 and N2 are splicing neutral and near neutral control sequences (Supplemental Fig. 2). U and D refer to upstream and downstream positions relative to the D1 5′ss. n.d. refers to not detected above background. All data were normalized to the constitutively spliced GH1 mRNA.

FIGURE 2.

MS2 tethering of splicing regulatory proteins mimics positional requirements for 5′ss activation. (A) Expression of MS2 fusion proteins results in position-dependent activation or repression of splicing using reporters that harbor MS2 binding sites upstream of or downstream from the D1 5′ss. Comparable expression of all MS2 fusion proteins was verified by Western blot analysis (Supplemental Fig. 5). (B) Schematic diagram of the splicing reporter in which the two MS2 binding sites are flanked by the identical up- and downstream 5′ss D1. The splicing pattern of each sample was analyzed using semi-quantitative RT-PCR analysis. (C) Identical experiments were performed using splicing reporters where the MS2 binding sites were replaced by high-affinity binding sites for endogenous TIA-1 and SRSF7.

FIGURE 3.

Binding of SRSF7 to the intron does not interfere with E-complex formation. (A) In vitro splicing reaction time course in the presence or absence of MS2-RS-SRSF7 or MS2-TIA-1 binding either upstream of or downstream from the 5′ss. (B) In vitro splicing reactions were repeated and quantified. (C) ATP-independent E-complex formation in the presence or absence of MS2-RS-SRSF7 or MS2-TIA-1 was visualized using agarose gel electrophoresis. (D) Higher-order, ATP-dependent complex formation in the presence or absence of MS2-RS-SRSF7 or MS2-TIA-1 was visualized using polyacrylamide native gels or agarose gels. Spliceosomal complexes or splicing products formed during the reactions are defined next to each gel.

FIGURE 4.

The recruitment of U1 snRNP components to the 5′ss is not decreased by repressive SREs. (A) RNAs were immobilized using Agarose beads and analyzed for the presence of U1 snRNP with specific antibodies directed against U1-70K and U1-C. MS2 coat protein was used as a loading control. N2 represents the splicing near neutral control sequence. (B) HeLa cell nuclear extracts were depleted (D) or mock depleted (U) of functional U1 snRNP using short DNA oligonucleotides and RNase H. The extracts were then used in RNA pulldown assays to demonstrate that U1 snRNA/RNA interactions are necessary for U1 snRNP recruitment. MS2 coat protein and hnRNP A1 were used to control for loading, and N2 represents splicing near neutral control sequences. (C) RNAs containing a muted 5′ss were used in the pulldown assay to demonstrate the effect of a functional or nonfunctional splice site on U1 recruitment.