Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 891-902 |
| Number of pages | 12 |
| Journal | Nucleic Acids Research |
| Volume | 37 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 24-07-2009 |
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All Science Journal Classification (ASJC) codes
- Genetics
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Isolation and characterization of post-splicing lariat-intron complexes. / Yoshimoto, Rei; Kataoka, Naoyuki; Okawa, Katsuya; Ohno, Mutsuhito.
In: Nucleic Acids Research, Vol. 37, No. 3, 24.07.2009, p. 891-902.Research output: Contribution to journal › Article
TY - JOUR
T1 - Isolation and characterization of post-splicing lariat-intron complexes
AU - Yoshimoto, Rei
AU - Kataoka, Naoyuki
AU - Okawa, Katsuya
AU - Ohno, Mutsuhito
PY - 2009/7/24
Y1 - 2009/7/24
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=63349111426&partnerID=8YFLogxK
U2 - 10.1093/nar/gkn1002
DO - 10.1093/nar/gkn1002
M3 - Article
C2 - 19103666
AN - SCOPUS:63349111426
VL - 37
SP - 891
EP - 902
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 3
ER -