The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation.
Because polyadenylation is essential for cell growth, in vivo examination of polyadenylation protein function has been difficult. Here we describe a new in vivo assay that allows structure-function assays on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. In this assay (the stem-loop luciferase assay for polyadenylation, SLAP), expression of a luciferase pre-mRNA with a modified downstream sequence element was made dependent upon co-expression of an MS2-CstF-64 fusion protein. We show here that SLAP accurately reflects CstF-64-dependent polyadenylation, confirming the validity of this assay. Using SLAP, we determined that CstF-64 domains involved in RNA binding, interaction with CstF-77 (the "Hinge" domain), and coupling to transcription are critical for polyadenylation. Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.
Hockert
J Andrew
JA
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6540, USA.
Yeh
Hsiang-Jui
HJ
MacDonald
Clinton C
CC
D000596:Amino Acids / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D039241:Cleavage Stimulation Factor / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D026723:Polyadenylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000596:Amino Acids; C474466:CSTF2T protein, human; C546606:CSTF3 protein, human; D039241:Cleavage Stimulation Factor; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012313:RNA; D008156:Luciferases