SR proteins are required for nematode trans-splicing in vitro.
SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts.
Sanford
J R
JR
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Bruzik
J P
JP
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D017164:Ascaris lumbricoides / Q000378:metabolism*; D002457:Cell Extracts; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
D002457:Cell Extracts; D011506:Proteins; D012322:RNA Precursors; D011994:Recombinant Proteins; D012694:Serine; D001120:Arginine