Interactions of CstF-64, CstF-77, and symplekin: implications on localisation and function.
Cleavage/polyadenylation of mRNAs and 3' processing of replication-dependent histone transcripts are both mediated by large complexes that share several protein components. Functional studies of these shared proteins are complicated by the cooperative binding of the individual subunits. For CstF-64, an additional difficulty is that symplekin and CstF-77 bind mutually exclusively to its hinge domain. Here we have identified CstF-64 and symplekin mutants that allowed us to distinguish between these interactions and to elucidate the role of CstF-64 in the two processing reactions. The interaction of CstF-64 with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation. In contrast, the nuclear accumulation of CstF-64 depends on its binding to CstF-77 and not to symplekin. Moreover, the CstF-64 paralogue CstF-64Tau can compensate for the loss of CstF-64. As CstF-64Tau has a lower affinity for CstF-77 than CstF-64 and is relatively unstable, it is the minor form. However, it may become up-regulated when the CstF-64 level decreases, which has biological implications for spermatogenesis and probably also for other regulatory events. Thus, the interactions between CstF-64/CstF-64Tau and CstF-77 are important for the maintenance of stoichiometric nuclear levels of the CstF complex components and for their intracellular localization, stability, and function.
Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland.
Marc-David
MD
Ruepp
Christoph
C
Schweingruber
Nicole
N
Kleinschmidt
Daniel
D
Schümperli
D026481:Blotting, Far-Western; D039241:Cleavage Stimulation Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026723:Polyadenylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D026721:RNA 3' End Processing; D039104:RNA 3' Polyadenylation Signals; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism*
D039241:Cleavage Stimulation Factor; D006657:Histones; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; C101043:SYMPK protein, human; D039221:mRNA Cleavage and Polyadenylation Factors