Nucleic Acids Research Advance Access originally published online on November 10, 2006
Nucleic Acids Research 2006 34(21):6264-6271; doi:10.1093/nar/gkl794
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Nucleic Acids Research, 2006, Vol. 34, No. 21 6264-6271
© 2006 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.
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Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs
1 Division of Systemic Life Science, Graduate School of Biostudies Kyoto University, Kyoto, Japan 2 Graduate School of Bioscience and Biotechnology Yokohama, Japan 3 Integrated Research Institute, Tokyo Institute of Technology Yokohama, Japan 4 Translational Research Center, Kyoto University Hospital Kyoto, Japan
*To whom correspondence should be addressed. Tel: 81 45 924 5872; Fax: 81 45 924 5834; Email: hhanda{at}bio.titech.ac.jp
Received July 31, 2006. Revised September 30, 2006. Accepted October 2, 2006.
Alternative polyadenylation leads to mRNAs with variable 3' ends. Since a 3'-untranslated region (3'-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3'-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3'-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3'-UTR. CFIm is a heterodimeric 3' end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3'-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.
Correspondence may also be addressed to Tadashi Wada. Email: tawada{at}bio.titech.ac.jp
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