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Nucleic Acids Research Advance Access published online on December 22, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn1002
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Isolation and characterization of post-splicing lariat–intron complexes

Rei Yoshimoto1, Naoyuki Kataoka1,2,*, Katsuya Okawa3 and Mutsuhito Ohno1,*

1Institute for Virus Research, Kyoto University, Kyoto, 606-8507, 2Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8510 and 3Frontier Technology Center, Graduate School of Medicine, Kyoto University, Kyoto, Japan

*To whom correspondence should be addressed. Tel: +81 3 5803 4877; Fax: +81 3 5803 5853; Email: kataoka.mtt{at}mri.tmd.ac.jp

Correspondence may also be addressed to Mutsuhito Ohno. Tel: +81 75 751 4018; Fax: +81 75 751-3992; Email: hitoohno{at}virus.kyoto-u.ac.jp

Received October 1, 2008. Revised November 27, 2008. Accepted November 30, 2008.

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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