Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A
- Rei Yoshimoto1,2,
- Daisuke Kaida1,3,4,
- Masaaki Furuno5,9,
- A. Maxwell Burroughs5,6,9,
- Shohei Noma5,
- Harukazu Suzuki5,9,
- Yumi Kawamura1,
- Yoshihide Hayashizaki7,9,
- Akila Mayeda2 and
- Minoru Yoshida1,8
- 1Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
- 2Division of Gene Expression Mechanism, Institute for Comprehensive Medical Science, Fujita Health University, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan
- 3Frontier Research Core for Life Sciences, University of Toyama, Toyama-shi, Toyama 930-0194, Japan
- 4Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama-shi, Toyama 930-0194, Japan
- 5RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
- 6National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA
- 7RIKEN Preventive Medicine and Diagnosis Innovation Program (PMI), Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
- 8Japan Science and Technology Corporation, CREST Research Project, Kawaguchi, Saitama 332-0012, Japan
- Corresponding authors: yoshidam{at}riken.jp, kaida{at}med.u-toyama.ac.jp
Abstract
Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5′ splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5′ splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.
Keywords
Footnotes
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↵9 These members of CLST and PMI belonged to RIKEN OSC before the RIKEN reorganization on April 1, 2013.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.058065.116.
- Received June 26, 2016.
- Accepted October 3, 2016.
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