The C-terminal domain of RNA polymerase II functions as a phosphorylation-dependent splicing activator in a heterologous protein.
RNA polymerase II, and specifically the C-terminal domain (CTD) of its largest subunit, has been demonstrated to play important roles in capping, splicing, and 3' processing of mRNA precursors. But how the CTD functions in these reactions, especially splicing, is not well understood. To address some of the basic questions concerning CTD function in splicing, we constructed and purified two fusion proteins, a protein in which the CTD is positioned at the C terminus of the splicing factor ASF/SF2 (ASF-CTD) and an RS domain deletion mutant protein (ASFDeltaRS-CTD). Significantly, compared to ASF/SF2, ASF-CTD increased the reaction rate during the early stages of splicing, detected as a 20- to 60-min decrease in splicing lag time depending on the pre-mRNA substrate. The increased splicing rate correlated with enhanced production of prespliceosomal complex A and the early spliceosomal complex B but, interestingly, not the very early ATP-independent complex E. Additional assays indicate that the RS domain and CTD perform distinct functions, as exemplified by our identification of an activity that cooperates only with the CTD. Dephosphorylated ASFDeltaRS-CTD and a glutathione S-transferase-CTD fusion protein were both inactive, suggesting that an RNA-targeting domain and CTD phosphorylation were necessary. Our results provide new insights into the mechanism by which the CTD functions in splicing.
Millhouse
Scott
S
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Manley
James L
JL
D000595:Amino Acid Sequence; D000818:Animals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D009687:Nuclear Proteins; D021122:Protein Subunits; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D012694:Serine; D012319:RNA Polymerase II