title: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
authors.forename: Andrei
authors.initials: A
authors.lastname: Alexandrov
authors.forename: David
authors.initials: D
authors.lastname: Colognori
authors.forename: Mei-Di
authors.initials: MD
authors.lastname: Shu
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry / Q000378:metabolism; D005091:Exons / Q000235:genetics*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D059365:Nonsense Mediated mRNA Decay / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D019696:Peptidylprolyl Isomerase; D011485:Protein Binding; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D039601:Eukaryotic Initiation Factor-4A; D039603:Eukaryotic Initiation Factor-4G; D009687:Nuclear Proteins; D019696:Peptidylprolyl Isomerase; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C578086:CWC22 protein, human; C500737:NOM1 protein, human