Isolation and characterization of post-splicing lariat-intron complexes.
Pre-mRNA splicing occurs in a large complex spliceosome. The steps of both spliceosome assembly and splicing reaction have been extensively analyzed, and many of the factors involved have been identified. However, the post-splicing intron turnover process, especially in vertebrates, remains to be examined. In this paper, we developed a two-tag affinity purification method for purifying lariat intron RNA-protein complexes obtained from an in vitro splicing reaction. Glycerol gradient sedimentation analyses revealed that there are at least two forms of post-splicing intron complexes, which we named the 'Intron Large (IL)' and the 'Intron Small (IS)' complexes. The IL complex contains U2, U5 and U6 snRNAs and other protein splicing factors, whereas the IS complex contains no such U snRNAs or proteins. We also showed that TFIP11, a human homolog of yeast Ntr1, is present in the IL complex and the TFIP11 mutant protein, which lacks the interaction domain with hPrp43 protein, caused accumulation of the IL complex and reduction of IS complex formation in vitro. Taken together, our results strongly suggest that TFIP11 in cooperation with hPrp43 mediates the transition from the IL complex to the IS complex, leading to efficient debranching and turnover of excised introns.
Yoshimoto
Rei
R
Institute for Virus Research, Kyoto University, Kyoto, 606-8507, Japan.
Kataoka
Naoyuki
N
Okawa
Katsuya
K
Ohno
Mutsuhito
M
D002499:Centrifugation, Density Gradient; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D009687:Nuclear Proteins / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification*
D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D012261:Ribonucleoproteins; C485860:TFIP11 protein, human; C539469:DHX15 protein, human; D020365:RNA Helicases