A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly.
Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF(65) and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5' splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.
Shen
Haihong
H
Howard Hughes Medical Institute, Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Green
Michael R
MR
D001120:Arginine / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D005091:Exons / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012322:RNA Precursors; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D012694:Serine; D001120:Arginine