Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly

Abstract

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.

Fig. 1.

Phosphorylation states of SRSF1 affect ESE binding affinity. (A) Cartoon representation of SRSF1 domain organization (upper) and coomassie stained SDS/PAGE showing unphosphorylated, hypo-phosphorylated (p-SRSF1), and hyper-phosphorylated SRSF1 (pp-SRSF1). (B and C) Filter binding assay showing the binding of SRSF1 (RRM1/2), SRSF1 (FL), p-SRSF1 (FL), and pp-SRSF1 (FL) to Ron ESE (AGGCGGAGGAAGC) and to mut Ron ESE (mRon ESE; AGGCGGUUGUUGC), respectively. The means and standard deviation (SD) of the results from three independent experiments are shown. (D) Estimated equilibrium dissociation constants (K_d) of SRSF1∶ESE complexes were measured based on FB assay (Ron^a) and EMSA (Ron^b) (see Fig. S1 A–C). K_d was estimated as 50% of bound RNA fraction. ND denotes not determined. SD was determined from three independent experiments.

Fig. 2.

Unphosphorylated RS domain interacts with ESE∶SRSF1 (RRM1/2). (A) EMSA showing the binding of SRSF1 (RRM1/2) to Ron ESE. (B) EMSA analysis of ESE mixed with GST-RS (197–248) and GST-pp-RS (197–248). (C) EMSA showing the binding of SRSF1 (RRM1/2) to GST-RS and GST-pp-RS. (D) GST pull-down assay showing binding between His-SRSF1 (RRM1/2) (2 μg) and GST-RS/GST-pp-RS (2 μg) in the absence or presence of Ron ESE or poly U₁₃. Input and bound proteins were detected by the Western blotting using anti-His Ab. Bound fractions was quantitated from three independent experiments (bottom). (E) The model depicting ESE binding to SRSF1 in its different phosphorylated states.

Fig. 3.

U1-70K binding by SRSF1. (A) Schematic representation of domain maps and fragments of U1-70K (left), phosphorylation mimetic versions of SRSF1 (right), and sequences and phosphorylated sites of RS1 and RS2 domains (bottom). (B) GST pull-down assay between different GST-SRSF1 constructs (10 μg) and 5 μl of in vitro transcribed-translated [³⁵S]-met labeled U1-70K constructs in the presence of RNase A (upper) or in the presence of cognate RNAs, Ron ESE and U1 snRNA (bottom). (C) Models depicting intra- and intermolecular binding modes within and between SRSF1 and U1-70K. (D) In vitro splicing of β-gb pre-mRNA in S100 complementation assay using WT and different phosphorylation mimics of SRSF1 (FL). Relative splicing efficiency of SRSF1 phosphomimetic mutants is shown in the bottom of the gel.

Fig. 4.

Two opposite surfaces of SRSF1 (RRMs) recruit ESE and U1-70K (RRM). (A) GST pull-down assay between GST-SRSF1 RRM1/2, RRM1, and RRM2 of 10 μg and U1-70K RRM (59–215) of 10 μg. (B) Ribbon presentation of SRSF1 (RRM1) (PDB ID code 1X4A). Putative RNA binding residues are denoted by yellow color. Residues in each mutant m1 (I32A/V35A), m2 (K38A/Y39A), and m3 (D68A/D69A) are denoted in three colors. (C) Ron ESE bindings to SRSF1 (RRM1/2) WT, m1, m2, m3, and FF-DD mutants (F56D/F58D) were measured by filter binding assay with error bars (SD) from three independent experiments. (D) Autoradiograph of GST pull-down assay between WT and mutants GST-SRSF1 (RRM1/2), and in vitro translated [³⁵S]-met labeled U1-70K (RRM). The binding fraction of the U1-70K (RRM) to WT and mutants GST-SRSF1 (RRM1/2) were quantitated from three independent experiments. (E) In vitro splicing of β-gb pre-mRNA in S100 complementation assay using WT and mutants SRSF1 (FL) and relative splicing efficiency is quantified as shown.

Fig. 5.

Dephosphorylated RS domain and U1-70K binding defective mutant of SRSF1 block early spliceosomal assembly steps. (A) Cartoon showing β-gb (Ron) and β-gb (Ron)ΔAG constructs. (B) Native gel analysis of the spliceosomal E complex formation by WT SRSF1 (RSRS) and different SRSF1 phosphomimetics in the presence of S100 extract and β-gb (Ron) pre-mRNA substrate. (C) Native gel analysis of the spliceosomal A and B/C complex formation of β-gb (Ron)ΔAG by SRSF1 phosphomimetics and WT (RSRS) without (left) or with 0.1 mg/mL tRNA (right) (D) In vitro splicing of the β-gb (Ron) pre-mRNA substrate by SRSF1 phosphomimetics in the presence of S100 extract. The relative splicing activities are shown at the bottom. (E) Native gel analysis of the E complex formation by WT and mutant SRSF1 (RRM1/2) in S100 extract. (F) Native gel analysis of the spliceosomal A and B/C complex formation of β-gb (Ron)ΔAG by WT and SRSF1 (RRM1/2) mutants without (left) or with 0.1 mg/mL tRNA (right) in S100 extract. (G) In vitro splicing assay of β-gb (Ron) pre-mRNA by WT and mutants SRSF1 (RRM1/2) in S100 extract. The relative splicing activities of SRSF1 (RRM1/2) mutants compared to WT as shown at bottom.

Fig. 6.

A model depicting the effect of RS phosphorylation in the E complex formation. Phosphorylation of the SRSF1 RS domain, mediated by the sequential actions of SRPK1 and CLK/STY, induces the dissociation of the RS from its RRM. Free SRSF1 (RRM) recruits U1 snRNP to the 5′SS through RRM-RRM interaction between SRSF1 and U1-70K. Released pp-RS domain interacts to the splicing factors bound to BPS/pY/3′SS to stabilize the E complex.