Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is shown to cooperate with the U1 snRNP particle in binding pre-mRNA. This cooperation is probably achieved by specific interactions between the arginine/serine-rich domain of the splicing factor and a similar region in a U1 snRNP-specific protein.

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The splicing factor U2AF (U2 snRNP auxiliary factor] is a heterodimer with subunits of 65 and 35 kD (U2AF^^ and U2AF^^). U2AF^^ binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF^^. Here, we report that U2AF^^ is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF^^ were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF^^. In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF^^ directly mediates interactions between U2AF^^ and proteins bound to the enhancers. Thus, U2AF^^ functions as a bridge between U2AF^^ and the enhancer complex to recruit U2AF^^ to the adjacent intron.[Key Words: U2AF; RNA; splicing; pre-mRNAs; SR proteins) Received March 1, 1996; revised version accepted April 22, 1996.The production of metazoan messenger RNAs (mRNAs) requires the accurate removal of introns from pre-messenger RNAs (pre-mRNAs) by RNA splicing (for review, see Moore et al. 1993). Initial recognition of the correct pairs of 5' and 3' splice sites by the splicing apparatus is a critical step in the processing of both constitutively and alternatively spliced pre-mRNAs (for review,

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5′ splice sites in a concentration‐dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP‐type RNA binding domains (RBDs) and a region highly enriched in repeating arginine‐serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains.