Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

doi:10.1083/jcb.143.2.297

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© The Rockefeller University Press, 0021-9525/1998//297 $5.00
The Journal of Cell Biology, Volume 143, Number 2, , 1998 297-307

Regular Articles

Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

Tom Misteli^*, Javier F. Cáceres, Jade Q. Clement, Adrian R. Krainer^*, Miles F. Wilkinson, and David L. Spector^*

^* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724; MRC Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom; and Department of Immunology, University of Texas M.D. Anderson Cancer Research Center, Houston, Texas 77030

Expression of most RNA polymerase II transcripts requires thecoordinated execution of transcription, splicing, and 3' processing.We have previously shown that upon transcriptional activationof a gene in vivo, pre-mRNA splicing factors are recruitedfrom nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription andpre-mRNA splicing within the cell nucleus. Here we have investigatedthe molecular basis for recruitment by analyzing the recruitmentproperties of mutant splicing factors. We show that multipleprotein domains are required for efficient recruitment of SRproteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exertdistinct functions in this process. In living cells, the RSdomain functions in the dissociation of the protein from speckles,and phosphorylation of serine residues in the RS domain isa prerequisite for this event. The RNA binding domains playa role in the association of splicing factors with the targetRNA. These observations identify a novel in vivo role for theRS domain of SR proteins and suggest a model in which proteinphosphorylation is instrumental for the recruitment of theseproteins to active sites of transcription in vivo.

Key Words: nucleus • phosphorylation • pre-mRNA splicing • recruitment • transcription

Abbreviations used in this paper: β-TM, β-tropomyosin; BKV, BK virus; CTD, COOH-terminal domain; GFP, green fluorescent protein; IGC, interchromatin granule cluster; PF, perichromatin fibril; Pol II, RNA polymerase II; PP1, protein phosphatase 1; RRM, RNA recognition motif.

T. Misteli dedicates this paper to the memory of Thomas Kreis.

T. Misteli was supported by the Human Frontiers Science Programand the Roche Research Foundation. A.R. Krainer was supportedby National Cancer Institute grant CA13106, M.F. Wilkinsonwas supported by National Institutes of Health (NIH) grant GM39586and National Science Foundation grant MCB-9307963, and D.L.Spector was supported by NIH grant GM42694.

Address all correspondence to D.L. Spector, Cold Spring HarborLaboratory, Cold Spring Harbor, NY 11724. Tel.: (516) 367-8456.Fax: (516) 367-8876. E-mail: spector{at}cshl.org

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