An evolutionarily conserved role for SRm160 in 3'-end processing that functions independently of exon junction complex formation.
SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.
McCracken
Susan
S
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Longman
Dasa
D
Johnstone
Iain L
IL
Cáceres
Javier F
JF
Blencowe
Benjamin J
BJ
D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000196:embryology / Q000235:genetics / Q000254:growth & development; D002460:Cell Line; D039241:Cleavage Stimulation Factor / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005091:Exons / Q000502:physiology*; D006801:Humans; D007163:Immunosorbent Techniques; D007438:Introns / Q000502:physiology; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology; D034622:RNA Interference / Q000502:physiology; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection
D034961:Antigens, Nuclear; D039241:Cleavage Stimulation Factor; D034521:Nuclear Matrix-Associated Proteins; D021122:Protein Subunits; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C111710:SRRM1 protein, human