Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs.
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.
Kubo
Tomohiro
T
Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Wada
Tadashi
T
Yamaguchi
Yuki
Y
Shimizu
Akira
A
Handa
Hiroshi
H
D020413:3' Untranslated Regions / Q000737:chemistry*; D004265:DNA Helicases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011061:Poly A / Q000032:analysis; D000075223:Poly-ADP-Ribose Binding Proteins; D026723:Polyadenylation; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D034622:RNA Interference; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D053669:Syndecan-2 / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics; D039221:mRNA Cleavage and Polyadenylation Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D020413:3' Untranslated Regions; D000075223:Poly-ADP-Ribose Binding Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; C450562:cleavage factor Im, human; D039221:mRNA Cleavage and Polyadenylation Factors; D019716:Tissue Inhibitor of Metalloproteinase-2; D053669:Syndecan-2; D011061:Poly A; D013762:Tetrahydrofolate Dehydrogenase; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes