Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1

Abstract

The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1's role in the mRNA's journey from splicing to decay has broad implications for gene expression regulation and genetic diseases.

Keywords: EJC; NMD; RNA splicing; SRSF1; UPF1.

Figure 1. SRSF1 Promotes NMD When Tethered Downstream of a PTC and Requires Its RRM2

(A) β-globin (HBB) wild-type (WT) and mutant (T39) reporter constructs without or with MS2-binding sites formed after splicing at the first (1MS2) or first and second exon-exon junctions (2MS2). Light gray ovals, PTCs; black ovals, stop codons. Diagrams are not to scale. (B) Representative western blot analysis of T7-tagged, T7-MS2-fused, and endogenous SRSF1, with β-catenin as a loading control. (C) Representative radioactive RT-PCR analysis of HeLa cells co-transfected with WT (W) or T39 (T) reporters, with (2MS2, gray bars) or without MS2- (white bars) binding sites, and T-SRSF1 or T-M-SRSF1 constructs. HBB bands were quantified, normalized to GFP, and plotted as T/W × 100 (mean and SEM) using GraphPad Prism. n = 3. (D) Representative radioactive RT-PCR analysis of U2OS cells co-transfected with HBB-WT or -T39 (T) reporters, with one MS2-binding site upstream of the T39 PTC (1MS2, light gray bars) or one MS2-binding site upstream and one downstream of PTC (2MS2, dark gray bars), and T-M-SRSF1 or empty vector control (T-MS2). Bands were quantified and plotted as above. n = 3. *p = 0.05 (Mann-Whitney). (E) Representative western blot probed with anti-T7 antibody, showing the expression of T-SRSF1 WT and deletion mutants. (F) Quantitation of radioactive RT-PCR of U2OS cells co-transfected with HBB-WT or Ter39 reporters and SRSF1 WT, deletion mutants, or T7-empty vector control. n = 3. (G) Representative western blot showing the expression of T-M-SRSF1 WT and deletion mutants. (H) Radioactive RT-PCR analysis as in (B) but with HBB-WT or Ter39 reporters with 2MS2-binding sites (2MS2) and T-MS2, T-M-SRSF1 WT, and deletion mutants. n = 3. Error bars represent SEM.

Figure 2. SRSF1 Co-immunoprecipitates with EJC Components and Splicing Enhances Its Effect on NMD

(A) Representative IP-western blot of nuclease-treated extracts from HeLa cells overexpressing T-SRSF1, T-ΔRRM2, or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous EJC components with the indicated antibodies. SRSF1 appears as several distinct bands due to its extensive phosphorylation (Hanamura et al., 1998). (B) Representative IP-western blot of nuclease-treated nuclear and cytoplasmic fractions from HeLa cells overexpressing T-SRSF1 or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous eIF4A3. LEDGF/p75 and Tubulin were used as nuclear and cytoplasmic markers, respectively. (C) Representative western blot showing siRNA-mediated knockdown of eiF4A3 (lane 2) and MAGOH (lane 3). β-catenin (β-cat) was used as a loading control. (D) Radioactive RT-PCR analysis of HeLa cells co-transfected with HBB-WT or Ter39 reporters and T-SRSF1 (+) or T7-empty vector control (−), in the presence of the indicated siRNAs. (E) HBB intronless cDNA WT and mutant (T39) reporter constructs without MS2-binding sites or with one MS2-binding site at the first (1/2M) or second (2/3M) exon-exon junction. Light gray ovals, PTCs; black ovals, stop codons. (F) Radioactive RT-PCR analysis of U2OS cells co-transfected with WT or Ter39 versions of HBB-cDNA, cDNA1/2M or cDNA2/3M reporters and T-M-SRSF1 (dark gray bars) or T-MS2 control (light gray bars). *p = 0.05 (Mann-Whitney). n = 3. (G) HBB with first or second intron deleted WT and mutant (T39) reporter constructs, with one MS2 binding site at the first (1/2M) or second (2/3M) exon-exon junction. Light-gray ovals: PTCs; black ovals: stop codons. (H) Radioactive RT-PCR analysis of HeLa cells co-transfected with WT or Ter39 versions of HBB-Δi1-1/2M or Δi2-2/3M reporters and T-M-SRSF1 (dark gray bars) or T-MS2 control (light gray bars). *p < 0.05 (Mann-Whitney). n = 3. Error bars represent SEM.

Figure 3. SRSF1/UPF3B Interaction Occurs in the Nucleus but Is Not Required to Stimulate NMD

(A) Representative IP-western blot of nuclease-treated HeLa cell extracts overexpressing T-SRSF1 WT, T-ΔRRM2 (Δ2), or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous UPF2 and UPF3B. (B) Representative IP-western blot of nuclease-treated nuclear (left panels) and cytoplasmic (right panels) fractions from HeLa cells overexpressing T-SRSF1 or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous UPF2 and UPF3B. Tubulin was used as a cytoplasmic marker. (C) Representative western blot showing siRNA-mediated knockdown of UPF2 (lane 2) and UPF3B (lane 3). β-catenin (β-cat) was used as a loading control. (D) Radioactive RT-PCR analysis of HeLa cells co-transfected with HBB-WT or Ter39 reporters and T-SRSF1 (+) or T7-empty vector control (−) in the presence of the indicated siRNAs. n = 3. Error bars represent SEM.

Figure 4. SRSF1 Interacts with UPF1 in the Nucleus and Cytoplasm and Enhances UPF1 Binding to mRNA

(A) Representative IP-western blot of nuclease-treated nuclear and cytoplasmic fractions from HeLa cells overexpressing T-SRSF1 or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous UPF1. Tubulin was used as a cytoplasmic marker. The interaction occurs in both the nucleus and cytoplasm (same samples were also used in Figure 2B). (B) IP-western blot analysis of HeLa cell extracts overexpressing T-SRSF1, T-SRSF1-NRS, or empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous UPF1 (first panel). The results show that a nuclear-retained form of SRSF1 interacts with UPF1. (C) Representative IP-western blot of HeLa cell extracts overexpressing FLAG-empty vector control or FLAG-UPF1, T-MS2 or T-MS2-SRSF1, and HBB-T39 or HBB-cDNAT39-2MS2, immunoprecipitated with anti-FLAG antibody and probed with the indicated antibodies. (D) Radioactive RT-PCR analysis of RNA extracted from the IPs shown in (B) of HBB-Ter39-2MS2 (light gray bars) and HBB-cDNATer39-2MS2 (dark gray bars). The ratio of IP/input was calculated and plotted. n = 3. (E) Representative IP-western blot of nuclear and cytoplasmic fractions from HeLa cells overexpressing FLAG-empty vector control or FLAG-SRSF1 and T7-empty vector control or T-SRSF1, immunoprecipitated with anti-FLAG antibody and probed for endogenous eIF4A3 and MAGOH. Tubulin was used as a cytoplasmic marker. (F) The intensities of bands corresponding to eIF4A3 (dark gray bars) and MAGOH (light gray bars) from both fractions were quantified and normalized first to input and then to IP efficiency, and the ratio of SRSF1+/SRSF1 was calculated. A ratio of 1 (line) indicates no change. n = 2. Error bars represent SEM.

Figure 5. SRSF1’s RRM2 Is Involved in UPF1 Dephosphorylation

(A) Representative IP-western blot analysis of nuclease-treated HeLa cell extracts overexpressing T-SRSF1 WT, T-ΔRRM2, or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed with the indicated antibodies. SRSF1 WT and ΔRRM2 co-immunoprecipitate with UPF1; however, a slower-migrating band increases in the ΔRRM2 IP (asterisk) and corresponds to p-T28-UPF1. (B) Representative IP-western blot of nuclease-treated extracts from HeLa cells overexpressing T-SRSF1 WT, T-ΔRRM2, or T7-empty vector control, immunoprecipitated with anti-T7 antibody and probed for endogenous SMG proteins with the indicated antibodies.

Figure 6. SRSF1 Promotes NMD of PTC-Containing Endogenous Transcripts

(A) Diagrams of four events that can introduce PTCs. White, PTC upon skipping of a cassette exon (Pan et al., 2006); light gray, PTC upon inclusion of a cassette exon (Pan et al., 2006); dark gray, splicing within the 3′ UTR; black, PTC introduced by a nonsense mutation (IDUA W402X; Scott et al., 1993). (B) Radioactive RT-PCR analysis of GFP-sorted HeLa cells (white and gray bars) or patient fibroblasts (black) co-transfected with T-SRSF1 or empty vector control and GFP. The data were plotted as the ratio of percent skipped or included mRNA from SRSF1-transfected cells over percent skipped or included mRNA from control transfections. The IDUA mRNA level was normalized to endogenous actin, and the mRNA ratio of SRSF1-transfected over control-transfected cells was plotted. n ≥ 2. (C) Same experimental design in HeLa cells as in (B) but expressing T-ΔRRM2. n = 2. A ratio of 1 (line) indicates no change in mRNA level between control and SRSF1 or ΔRRM2 overexpression. Arrows indicate true NMD targets, sensitive to cycloheximide treatment. See also Figure S7. Error bars represent SEM.

Figure 7. SRSF1’s Functions in NMD

(A) A simplified diagram of the NMD pathway is shown on the left. On the right, SRSF1 recruits UPF1 and interacts with the EJC and UPF3B in the nucleus. As the mRNP complex is exported to the cytoplasm, translation occurs in the presence of UPF1, bypassing UPF2 recruitment and initiating NMD. (B) On the left, SRSF1 assists in p-UPF1 dephosphorylation and release from the transcript via interactions with p-UPF1, SMG7, and PP2A (Michlewski et al., 2008). On the right, SRSF1 lacking RRM2 reduces the interaction between the RRM2 mutant and SMG7, decreasing PP2A-mediated dephos-phorylation of UPF1 and leading to a hyperphosphorylated form of UPF1. P, PTC; T, termination codon; green Y, Y14; green M, MAGOH; red Ps, phosphorylated residues in UPF1; orange line, nuclear membrane.