Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing

Abstract

The conserved pre-mRNA splicing factor SF1 is implicated in 3' splice site recognition by binding directly to the intron branch site. However, because SF1 is not essential for constitutive splicing, its role in pre-mRNA processing has remained mysterious. Here, we used crosslinking and immunoprecipitation (CLIP) to analyze short RNAs directly bound by human SF1 in vivo. SF1 bound mainly pre-mRNAs, with 77% of target sites in introns. Binding to target RNAs in vitro was dependent on the newly defined SF1 binding motif ACUNAC, strongly resembling human branch sites. Surprisingly, the majority of SF1 binding sites did not map to the expected position near 3' splice sites. Instead, target sites were distributed throughout introns, and a smaller but significant fraction occurred in exons within coding and untranslated regions. These data suggest a more complex role for SF1 in splicing regulation. Indeed, SF1 silencing affected alternative splicing of endogenous transcripts, establishing a previously unexpected role for SF1 and branch site-like sequences in splice site selection.

Figure 1.

Isolation of SF1–RNA complexes from crosslinked HeLa cells. (A) Lysates of crosslinked HeLa cells (lane 1) were incubated with Dynabeads in the presence (lane 2) or absence (lane 3) of mAb24D1. Input (lane 1) and unbound material (lanes 2 and 3) were separated by 10% SDS–PAGE. After transfer to nitrocellulose SF1 was detected with mAb24D1. Protein size markers (in kDa) are indicated on the left. (B) SF1–RNA complexes eluted from mAb24D1-coupled Dynabeads were separated by 10% Bis–Tris NuPAGE and visualized by autoradiography. Protein size markers are shown on the left. Gel slices used for RNA analysis in panel C are marked on the right. (C) RNA extracted from nitrocellulose slices 1–5 in panel B was resolved in a denaturing 13% polyacrylamide gel. DNA size markers (in bp) are shown on the left. (D) Pie chart representing the number of SF1 CLIP tags in the protein-coding genes, genes encoding ncRNAs and in intergenic regions. (E) Pie chart representing the number of SF1 CLIP tags in introns or exons of protein-coding genes. Exonic CLIP tags were further divided into coding sequences (CDS), 5′- and 3′-UTRs and exon–exon junctions.

Figure 2.

Determination of the optimal SF1–RNA-binding motif. (A) In vitro-transcribed RNAs containing the wild-type or mutant BPS were incubated with buffer or SF1-KH/QUA2 (5, 10 and 20 µM; indicated by triangles above the figure). Reaction products were separated by native PAGE and visualized by autoradiography. The same wild-type images were used for positions –5/–4/–3, –2/+1 and BP/–1. (B) Web logo representing the weight matrix for each position of the SF1 binding site derived from the quantification of the data presented in A.

Figure 3.

Cooperative binding of SF1 and U2AF65 to an endogenous SF1 target. (A) Scheme of SF1 and U2AF65 constructs used for EMSA. The U2AF65 interaction domain (U2AF65-ID), KH/QUA2 domain and the zinc knuckle (Zn) of SF1 are shown, as well as the arginine/serine-rich (RS) domain, RRMs 1 and 2 and the UHM of U2AF65. The star above the U2AF65-ID of SF1-C4 indicates the location of the W22A mutation. Numbers indicate amino acids of the truncated proteins. Variability in the length of SF1 isoforms is indicated by dashed lines. (B) RNAs corresponding to CLIP tag 2-50 (wild-type, WT, or mutants M1–M6) were transcribed in vitro. SF1 binding motifs are indicated in bold. Mutations are underlined. (C) Wild-type and mutant RNAs were incubated with buffer or SF1-KH/QUA2 (5, 10 and 20 µM; indicated by triangles). Reaction products were separated by native PAGE and visualized by autoradiography. (D) RNA 2–50 WT was incubated with buffer or U2AF65Δ1–94 (0.2, 0.5, 1, 2 and 4 µM; indicated by triangles) in the absence or presence of 6.6 µM SF1-C4 as indicated. Reaction products were separated by native PAGE and visualized by autoradiography. The migration of RNA 2–50 bound to U2AF65Δ1–94 (open arrowheads), SF1-C4 (vertical bar) or both (closed arrowheads) is indicated.

Figure 4.

Cooperative binding of SF1 and U2AF65 to an endogenous SF1 target with a suboptimal SF1 binding site. (A) RNAs corresponding to wild-type (WT) and mutant (M) CLIP tag 1–10 were transcribed in vitro. A SF1 binding motif is indicated in bold. The mutation is underlined. (B) Wild-type and mutant RNAs were assayed as in Figure 3C. (C) Wild-type and mutant RNAs were tested as in Figure 3D in the absence or presence of 6.6 µM SF1-C4 or SF1-C4/W22A as indicated. The migration of RNAs bound to U2AF65Δ1–94 (open arrowheads), SF1-C4 (vertical bar) or both (closed arrowheads) is indicated.

Figure 5.

Effects of RNAi-mediated depletion of SF1 on the splicing of CLIP tag-containing pre-mRNAs. (A) cDNA from HeLa cells transfected in the absence (mock) or presence of siRNAs targeting SF1 (SF1 #1 and #2), luciferase (LUC) or SF3a120, as indicated on top of each panel, was PCR-amplified with primers specific for SF1, SF3a120 and H3F1 mRNAs, products were separated by PAGE and visualized by autoradiography. The numbers below the ‘mRNA’ panels indicate the percentage of mRNA normalized to the LUC control. Data represent the average of two experiments; the standard deviations were <1. HeLa cell lysates prepared in parallel were separated by SDS–PAGE and proteins revealed by western blotting. (B, C, D and E) The effect of SF1 depletion on the splicing of endogenous FGFR1OP (B), TNIP1 (C), PLOD2 (D) and UPF3A (E) pre-mRNAs was analyzed as in panel A. Sizes of DNA markers and spliced products are shown to the left and right of the images, respectively. Results of at least two experiments were quantified and are expressed as percent exon inclusion below the panels in addition to the standard deviation (std. dev.). The asterisk (panel B) indicates a PCR product that could not be identified by sequencing. The schemes depict pre-mRNA regions subject to alternative splicing (not to scale) and observed splicing patterns. Forward (F) and reverse (R) PCR primers including the exon number (E#) are shown above the schemes. Filled circles indicate the approximate location of CLIP tags. TNIP exon 2 is transcribed from an alternative promoter and not included in mRNAs transcribed from exon 1.