Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins.
During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.
Lindstrom
D L
DL
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064, USA.
Squazzo
S L
SL
Muster
N
N
Burckin
T A
TA
Wachter
K C
KC
Emigh
C A
CA
McCleery
J A
JA
Yates
J R
JR
Hartzog
G A
GA
D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D056488:Histone Chaperones; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D012322:RNA Precursors; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D035165:Transcription Factors, General / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000302:isolation & purification / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000098556:mRNA Guanylyltransferases
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D056488:Histone Chaperones; C471552:IWS1 protein, S cerevisiae; D009687:Nuclear Proteins; C106768:POB3 protein, S cerevisiae; D012322:RNA Precursors; D012333:RNA, Messenger; C071238:SPT16 protein, S cerevisiae; C080991:SPT4 protein, S cerevisiae; C065456:SPT6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D035165:Transcription Factors, General; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; C069940:SPT5 transcriptional elongation factor; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D012319:RNA Polymerase II; D000098556:mRNA Guanylyltransferases; C056604:transcription factor TFIIF