Pre-mRNA 3' cleavage is reversibly inhibited in vitro by cleavage factor dephosphorylation.
During 3' end formation most pre-mRNAs undergo endonucleolytic cleavage and polyadenylation in the 3' untranslated region. Very little is known concerning the role that post-translational modifications play in the function and regulation of the factors required for 3' cleavage. Using the reconstituted pre-mRNA cleavage reaction, we find that non-specific dephosphorylation of HeLa cell nuclear extract leads to the loss of 3' cleavage activity. A variety of serine/threonine phosphatases inhibited cleavage activity, while a tyrosine phosphatase did not. When the three major cleavage factor activities-CPSF, CstF and CF(m) (containing CFI(m) and CFII(m))-were separated and dephosphorylated individually, only CF(m) was found to lose activity, indicating that the target of dephosphorylation resides within this fraction. In accordance with this result, only CF(m) was able to restore cleavage activity to HeLa nuclear extract whose 3' cleavage activity had been completely inactivated by dephosphorylation. We conclude that at least one subunit of either CFI(m) or CFII(m) requires serine or threonine phosphorylation to function during 3' cleavage. Our data suggest that cleavage factor phosphorylation may serve as a regulatory on/off switch to control pre-mRNA 3' end formation.
Ryan
Kevin
K
Department of Chemistry, City College of New York, New York, New York 10031 USA. kr107@sci.ccny.cuny.edu
D020413:3' Untranslated Regions; D002848:Chromatography, DEAE-Cellulose; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D010766:Phosphorylation; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D020413:3' Untranslated Regions; D012322:RNA Precursors; D012333:RNA, Messenger; D013912:Threonine; D012694:Serine