U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

Abstract

A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5' splice site of E4 is coincident with the selection of the 3' splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3' splice site of E4 in a manner that is clearly facilitated by a downstream 5' splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3' splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.[Key Words: RNA-protein binding; U1 snRNP; U2AF65; alternative splicing; exon selection] Received May 8, 1992; revised version accepted October 2, 1992.Alternative splicing of pre-mRNA is a widespread process that is largely responsible for the diversity of polypeptides expressed in mammalian cells, yet a molecular understanding of the mechanisms and factors that govern this process is lacking. A central question of interest is how to explain the regulation of splice site selection that leads to the observed patterns of alternative 5', or 3' splice site selection, mutually exclusive exon selection, or controlled exon skipping (Andreadis et al. 1987;Maniatis 1991).Our current understanding, from studies of single iniron-containing pre-mRNA substrates, is that splice site recognition is achieved by the assembly of a spliceosome complex. The spliceosome is known to contain the small nuclear ribonucleoprotein (snRNP) particles U1, U2, U5, and U4 + U6, as well as additional protein factors Steitz et al. 1988;Green 1991;Guthrie 1991). In one of the earliest spliceosome assembly events, the 5' splice site at the upstream boundary of the intron is recognized by direct base-pairing with a 6-to 9-nucleotide stretch at the 5' end of U1 small nuclear ~Affifiated with btCB Graduate Program, Brown University, Providence, Rhode Island 02912 USA. 2Corresponding author.RNA (snRNA) (Zhuang and Weiner 1986;Siliciano and Guthrie 1988;Rosbash and Seraphin 1991). This RNA base-pairing interaction appears to be further stabilized by the involvement of a U1 snRNP-specific protein, U1-C (Heinrichs et al. 1990). Interestingly, engineered nucleotide changes in the 5' end of U1 snRNA have been shown to alter 5' splice s...

Splicing inhibition of U2AF 65 leads to alternative exon skipping

“…In alternative splicing, U2AF 65 has been reported to be a regulatory target of multiple splicing factors (30,31), but its role in alternative splicing has not been clear. From systematic evolution of ligands by exponential enrichment (SELEX) and RNA seq data, it was demonstrated that U2AF 65 has a high specificity for CU-rich sequences and that U2AF 65 specifically contacts Py tract in vivo (10,(39)(40)(41) It has not been reported that U2AF 65 interacts with enhancers or inhibitors on pre-mRNA, which is different from SR proteins and hnRNP, which interact with enhancer or inhibitor sequences on pre-mRNA. It was shown in previous reports that increased binding of U2AF 65 is directly related with increased splicing activity (31,42).…”

“…Using U2AF 65 depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF 65 is an essential splicing factor (9). Whereas U2AF 65 binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF 35 plays a role in the 3′ splice-site (10,11). As U2AF 65 prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF 65 and Py tract promotes prespliceosome assembly (12).…”

Functional selection of splicing enhancers that stimulate trans-splicing in vitro

“…In addition to the classic role of U1 snRNP in base pairing to 59 splice sites as an initial event in the cissplicing pathway, other functions have also been ascribed+ These include the 59 splice site-independent interaction between U1 and U2 (Zillmann et al+, 1987;Barabino et al+, 1990;Fu & Maniatis, 1992)+ This interplay was further determined to be refractory to either RNase H-targeted removal of the 59 end of U1 (Fu & Maniatis, 1992) or blockage of this same region with a 29-O-methyl oligonucleotide (Barabino et al+, 1990)+ In fact, participation of U1 snRNP in some aspect of premRNA splicing, other than base pairing to the 59 splice site, was demonstrated through the use of nonstandard substrates (Seiwert & Steitz, 1993)+ Perhaps the most intriguing of the proposed roles of U1 in pre-mRNA splicing relates to the ability of U1 snRNP to promote splicing of upstream introns through exon definition (Robberson et al+, 1990)+ Direct interaction of the 59 end of U1 snRNA was shown to mediate this effect (Talerico & Berget, 1990;Kreivi et al+, 1991;Kuo et al+, 1991)+ In this case, U1 is able to positively effect the splicing of an upstream intron through promotion of complex formation on the 39 splice site of the preceding intron (Hoffman & Grabowski, 1992;Chiara & Reed, 1995)+ It has been suggested that this role for U1 might actually indicate its major function (Cohen et al+, 1994;Hwang & Cohen, 1996)+ In transsplicing, where there is no required base pairing interaction between the 59 splice site and U1 (Hannon et al+, 1991), we have demonstrated that U1 interaction with a sequence downstream of the 39 trans acceptor can positively affect the reaction+ Due to the fact that the 59 splice site in the trans-splicing reaction is contained on a single SL RNP in A. lumbricoides, there is no possibility of differences in the sequence of the 59 splice site that might allow for regulation of trans-splicing+ The inclusion of discrete splicing enhancers or 59 splice sites downstream of the trans-spliced 39 splice site might allow for some level of trans-splicing regulation+ This would be especially important in cases where there is a decision between cis-and trans-splicing as in the case of the fert-1 gene (Spicher et al+, 1994)+ In addition, the fact that 59 splice sites normally follow 39 trans acceptors in nematodes may allow for exon definition to actively promote constitutive trans-splicing, within distance constraints, in a large number of transcripts+…”

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

“…We subsequently investigated the movement of various mutant (altered or truncated) pre-mRNA to speckles, and also examined the effects of the second exon and intron, which are known for their role in the prespliceosome assembly (Hoffman and Grabowski, 1992;Staknis and Reed, 1994). Biochemical studies have suggested that the formation of the prespliceosome complex was blocked and that the kinetics of the splicing process were thus influenced at different stages of the splicing reaction.…”