Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay

Abstract

The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the nonsense-mediated decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association. In vivo depletion of hCWC22, as for yeast Cwc22p, causes a splicing defect, resulting in decreased levels of mature cellular mRNAs. Nonetheless, hCWC22 depletion yields increased levels of spliced RNA from the unusual nonsense codon-containing U22 host gene, which is a natural substrate of NMD. To test whether hCWC22 acts in NMD through coupling splicing to EJC deposition, we searched for mutations in hCWC22 that affect eIF4AIII deposition without affecting splicing. Addition of hCWC22(G168Y) with a mutation at the putative hCWC22/eIF4AIII interface exacerbates the defect in splicing-dependent deposition of eIF4AIII(T334V) with a mutation reported to be in direct contact with mRNA, linking hCWC22 to the process of EJC deposition in vitro. Importantly, the addition of hCWC22(G168Y) affects deposition of eIF4AIII(T334V) without inhibiting splicing or the efficiency of deposition of the endogenous eF4AIII(WT) in the same reaction, demonstrating hCWC22's specific role in eIF4AIII deposition in addition to its role in splicing. The essential splicing factor CWC22 has, therefore, acquired functions in EJC assembly and NMD during evolution from single-celled to complex eukaryotes.

Fig. 1.

hCWC22 directly interacts with eIF4AIII in vitro and in vivo; mutations in the MIF4G domain of hCWC22 disrupt the interaction. (A) eIF4AIII-binding sites of CWC22 and NOM1 are more homologous to each other than to the eIF4A-binding sites of eIF4G or death-associated protein 5 (DAP5) as seen from the alignment of their major (12-aa motif) and minor (second motif) binding sites. Positions in eIF4G that directly contact eIF4A in the yeast crystal structure (21) are indicated with black dots. Positions in Sgd1p that resulted in (i) allele-specific suppression and (ii) synthetic interaction with fal1(T322V) (16) are indicated with a triangle and diamond, respectively. (B) Structure of the yeast eIF4G/eIF4A complex (21); binding surfaces are outlined with red ovals. Amino acids that make direct contacts and were used for alanine substitutions in this work are underlined. (C) GST pull-down shows direct physical interaction between human eIF4AIII and hCWC22 recombinant proteins. The black arrow indicates the Coomassie-stained hCWC22 band copurifying with GST-eIF4AIII. The white arrow shows the absence of detectable hCWC22 copurifying with GST-Trm82p, which provides a negative control. GST-eIF4AIII and GST-Trm82p were expressed and purified from yeast, whereas hCWC22 was expressed and purified from E. coli. I1 and I2 denote inputs (100%) for pull-downs shown in lanes 1 and 2 and lanes 3 and 4, respectively. (D) Human eIF4AIII and hCWC22 coimmunoprecipitate from cell extracts; mutations in the MIF4G domain of hCWC22 disrupt the interaction. The Western blot shows anti-FLAG immunoprecipitates from nuclear extracts of HEK293T cells transiently transfected with plasmids expressing CMV-driven tagged versions of eIF4III, hCWC22, and a negative control protein METTL1, as indicated. The IP reactions were performed in the presence of RNase A (Qiagen) as described in the SI Materials and Methods. The FLAG and c-Myc epitopes were detected using mouse monoclonal M2 and HRP-conjugated mouse monoclonal 9E10 antibodies, respectively.

Fig. 2.

Knockdown of hCWC22 decreases levels of spliced cellular mRNAs but increases levels of spliced RNA of UHG, a natural NMD substrate. (A) The Western blot shows the level of hCWC22 in HeLa cells (lane 1) and the results of siRNA-mediated knockdown of hCWC22 without (lane 2) or with expression (lane 3) of its siRNA-resistant variant; α-tubulin provides a loading control. (B) RT-qPCR analysis of the levels of spliced GAPDH, B2M, and HPRT1 mRNAs in the cells used in A. (C) Schematic representation of the positions of starting methionines (Met) and PTCs (shown as stop signs) in the UHG(WT), an NMD-resistant mutant variant UHG(NMD^R), and the NMD-resistant variants into which a single premature stop codon (PTC1 or PTC3) was reintroduced [the 19 stop codons in the last (11th) exon are not expected to elicit NMD]. (D) Human UHG is an efficient NMD substrate. RT-qPCR analysis of spliced transcripts from the WT and mutant UHG constructs diagramed in F was performed with primers complementary to the CMV promoter-specific 5′-untranslated sequence (UHG_q1F) and exon 3 of UHG (UHG_q2R). The resulting qPCR products were gel-purified and sequenced to ensure their identity as spliced UHG RNA with introns 1 (399 nt) and 2 (207 nt) removed. Levels of cotransfected GFP (pmaxGFP) served as a control for transfection efficiency. (E) RT-qPCR analysis of the levels of spliced endogenous UHG RNA in the cells used in A reveals a marked increase upon hCWC22 knockdown. qPCR primers were complementary to exon 2 (UHG1F) and exon 3 (UHG1R) (set 1) or to the junction of exons 9 and 10 (UHG3F) and exon 11 (UHG3R) (set 2).

Fig. 3.

Synthetic effect of mutations hCWC22(G168Y) and eIF4AIII(T304V) results in decreased EJC deposition on AdML RNA without affecting splicing in vitro. (A) Schematic of the experiment [based on the EJIPT assay (25)] that simultaneously detects (i) the efficiency of deposition of FLAG-tagged eIF4AIII variants on spliced biotinylated AdML RNA and (ii) the efficiency of splicing in the same reaction. Whereas the deposition efficiency of FLAG-tagged eIF4AIII variants on biotinylated RNA differs, the equivalent deposition of endogenous eIF4AIII provides a sensitive measure of splicing. (B) Western blots show FLAG-tagged and endogenous eIF4AIII coprecipitated with biotinylated AdML RNA after in vitro splicing at 20 °C for 12 h. HeLa nuclear extract (60 μL) (33) was supplemented with 25 μL each of two HEK293T whole-cell extracts (34) expressing one FLAG-eIF4AIII, one hCWC22 variant, or a control green fluorescent protein (−), as indicated. Intronless biotinylated AdML mRNA substrate was used in place of the AdML pre-mRNA in lane 10 (which was otherwise identical to lane 8) to determine the background level of splicing-independent coprecipitation. Total eIF4AIII and the FLAG-tagged eIF4AIII were detected as described in Materials and Methods. (C) The bar graph summarizes quantification of three blots as shown in B; quantification of individual blots (Fig. S5) showed the synthetic effect in each experiment.