The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export.
Spt6 promotes transcription elongation at many genes and functions as a histone H3 chaperone to alter chromatin structure during transcription. We show here that mammalian Spt6 binds Ser2-phosphorylated (Ser2P) RNA polymerase II (RNAPII) through a primitive SH2 domain, which recognizes phosphoserine rather than phosphotyrosine residues. Surprisingly, a point mutation in the Spt6 SH2 domain (R1358K) blocked binding to RNAPIIo without affecting transcription elongation rates in vitro. However, HIV-1 and c-myc RNAs formed in cells expressing the mutant Spt6 protein were longer than normal and contained splicing defects. Ectopic expression of the wild-type, but not mutant, Spt6 SH2 domain, caused bulk poly(A)+ RNAs to be retained in the nucleus, further suggesting a widespread role for Spt6 in mRNA processing or assembly of export-competent mRNP particles. We cloned the human Spt6-interacting protein, hIws1 (interacts with Spt6), and found that it associates with the nuclear RNA export factor, REF1/Aly. Depletion of endogenous hIws1 resulted in mRNA processing defects, lower levels of REF1/Aly at the c-myc gene, and nuclear retention of bulk HeLa poly(A)+ RNAs in vivo. Thus binding of Spt6 to Ser2-P RNAPII provides a cotranscriptional mechanism to recruit Iws1, REF1/Aly, and associated mRNA processing, surveillance, and export factors to responsive genes.
Yoh
Sunnie M
SM
Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Cho
Helen
H
Pickle
Loni
L
Evans
Ronald M
RM
Jones
Katherine A
KA
D019943:Amino Acid Substitution; D000818:Animals; D026481:Blotting, Far-Western; D002467:Cell Nucleus / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D005095:Exoribonucleases / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D010768:Phosphoserine; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors; D015533:Transcriptional Activation; D018909:src Homology Domains
C510636:Iws1 protein, human; D009687:Nuclear Proteins; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C502250:Supt6 protein, mouse; D014157:Transcription Factors; D010768:Phosphoserine; D012319:RNA Polymerase II; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C077044:EXOSC10 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase