Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing.
The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.
Wang
J
J
Department of Biological Sciences, Columbia University, New York, New York 10027 USA.
Xiao
S H
SH
Manley
J L
JL
D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D015696:Gene Products, tat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics*; D012322:RNA Precursors; D012326:RNA Splicing; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000068103:Serine-Arginine Splicing Factors