SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo.
The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.
Sapra
Aparna K
AK
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany.
Ankö
Minna-Liisa
ML
Grishina
Inna
I
Lorenz
Mike
M
Pabis
Marta
M
Poser
Ina
I
Rollins
Jarod
J
Weiland
Eva-Maria
EM
Neugebauer
Karla M
KM
D047369:Chromatin Immunoprecipitation; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D016762:Genes, fos; D049452:Green Fluorescent Proteins / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012326:RNA Splicing / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012261:Ribonucleoproteins / Q000096:biosynthesis* / Q000378:metabolism; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D049452:Green Fluorescent Proteins