Multiple roles for SR proteins in trans splicing.
The trans-splicing reaction involves the association of 5' and 3' splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3' acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5' splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.
Furuyama
Suzanne
S
Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Bruzik
James P
JP
D000818:Animals; D017164:Ascaris lumbricoides; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D023282:Nuclease Protection Assays; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D012326:RNA Splicing / Q000502:physiology*; D020038:RNA, Spliced Leader / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes
D036022:Capsid Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020038:RNA, Spliced Leader; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D000068103:Serine-Arginine Splicing Factors