Functional coupling of RNAP II transcription to spliceosome assembly.
The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent pre-messenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.
Das
Rita
R
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Dufu
Kobina
K
Romney
Ben
B
Feldt
Megan
M
Elenko
Mark
M
Reed
Robin
R
D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000502:physiology*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors; D012261:Ribonucleoproteins; D014764:Viral Proteins; D012319:RNA Polymerase II; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases