Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing

Abstract

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, U2AF 65 , 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.

Axis inhibition protein 2 (AXIN2) polymorphisms may be a risk factor for selective tooth agenesis

“…SC35 and SF2/ASF proteins have been found in the pre-spliceosomal E complex and have also been implicated in many aspects of constitutive and regulated splicing (Blencowe 2000). The SC35 motif can promote the splicing process, whereas the SF2/ASF protein can repress it (Wang et al 1998;Liu et al 2000). Thus, we suggest that replacement of the SC35 binding site by SF2/ASF -following c.2062C>T transition -may negatively affect splicing efficiency and the cellular concentration of AXIN2 and lead to abnormalities in tooth development (Fig.…”

The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers

“…The role of the different SR protein domains in the constitutive splicing reaction, alternative splicing, and general interactions with RNA has been analyzed by different means+ The RS domain is absolutely required for constitutive splicing (Cáceres & Krainer, 1993;Zuo & Manley, 1993;Wang et al+, 1996) and functions primarily in protein-protein interactions with other SR proteins or SR protein-related polypeptides such as U1-70K or U2AF35 (Wu & Maniatis, 1993;Kohtz et al+, 1994;Xiao & Manley, 1997)+ In addition, the RS domains of ASF/SF2 and some other SR proteins appear to be functionally interchangeable in substrate commitment assays (Chandler et al+, 1997) and in a cell viability system (Wang et al+, 1998) and those of the major SRp30s may function more or less equivalently as splicing activators, independently of the RNA binding domain (Graveley & Maniatis, 1998)+ In contrast, it seems more and more clear that SR protein-specific functions rely upon their ability to interact specifically with the pre-mRNA sequences+ Until recently, however, only limited details were available concerning these interactions+ Using a SELEX approach, RNA targets have been identified for ASF/SF2 and SC35 (Tacke & Manley, 1995), RBP1 (Heinrichs & Baker, 1995) and more recently for SRp40 (Tacke et al+, 1997) and SRp55 (Shi et al+, 1997)+ Unfortunately, except for SRp40, which can be easily distinguished from other SR species (Tacke et al+, 1997), it remains unclear whether or not individual SR proteins, in the context of a nuclear extract, still efficiently and preferentially recognize their specific target RNA sequences+…”

Processive phosphorylation: Mechanism and biological importance

“…For SRPK1, this substrate specificity is governed by a docking motif N-terminal to the RS domains, which restricts the number of serines in ASF/SF2 that can be phosphorylated [74] (Figure 2). In the cell, SRPK1 is found in the cytoplasm, while Clk/Sty is localized in the nucleus [66,[75][76][77]. The current model is that SRPK1 phosphorylates ASF/SF2 in the cytoplasm, triggering nuclear entry and recruitment to speckles.…”