The Carboxyl-terminal Domain of RNA Polymerase II Is Not Sufficient to Enhance the Efficiency of Pre-mRNA Capping or Splicing in the Context of a Different Polymerase.
Eukaryotic messenger RNA precursors (pre-mRNAs) synthesized by RNA polymerase II (RNAP II) are processed co-transcriptionally. The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to mediate the coupling of transcription with pre-mRNA processing by coordinating the recruitment of processing factors during synthesis of nascent transcripts. Previous studies have demonstrated that the phosphorylated CTD is required for efficient co-transcriptional processing. In the study presented here we investigated whether the CTD is sufficient to coordinate transcription with pre-mRNA capping and splicing in the context of two other DNA-dependent RNA polymerases, mammalian RNAP III and bacteriophage T7 RNAP. Our results indicate that the CTD fused to the largest subunit of RNAP III (POLR3A) is not sufficient to enhance co-transcriptional pre-mRNA splicing or capping in vivo. Additionally, we analyzed a T7 RNAP-CTD fusion protein and examined its ability to enhance pre-mRNA splicing and capping of both constitutively and alternatively spliced substrates. We observed that the CTD in the context of T7 RNAP was not sufficient to enhance pre-mRNA splicing or capping either in vitro or in vivo. We propose that the efficient coupling of transcription to pre-mRNA processing requires not only the phosphorylated CTD but also other RNAP II specific subunits or associated factors.
Natalizio
Barbara J
BJ
Department of Molecular Genetics and Microbiology, Center for RNA Biology, and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Robson-Dixon
Nicole D
ND
Garcia-Blanco
Mariano A
MA
D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary / Q000502:physiology; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D012315:RNA Caps; D014764:Viral Proteins; D012319:RNA Polymerase II; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C585825:POLR3A protein, human; D012320:RNA Polymerase III