Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF³⁵ and for Its Function after Polypyrimidine Tract Recognition by U2AF⁶⁵

Abstract

35 and a novel function for this factor in pre-mRNA splicing.The first ATP-dependent step in the assembly of splicing complexes is the stable association of U2 snRNP with the 3Ј part of the intron (reviewed in reference 12), which includes the branch point region, the polypyrimidine (Py) tract, and the conserved dinucleotide AG at the 3Ј splice site. The branch point region establishes base-pairing interactions with U2 snRNA that are critical for catalysis of the splicing reaction. The Py tract, particularly important in higher eukaryotes, is a pyrimidine-rich sequence located between the branch point and the AG dinucleotide that serves as the binding site for the U2 snRNP auxiliary factor (U2AF).Human U2AF is an essential splicing factor purified as a heterodimer composed of 65-kDa (U2AF 65 ) and 35-kDa (U2AF 35 ) subunits (39). U2AF 65 binds directly to Py tracts (41), while U2AF 35 is tethered to the pre-mRNA through its interaction with U2AF 65 (42). When bound to the Py tract, the amino-terminal arginine-serine-rich (RS) domain of U2AF 65 contacts the branch point region, and it has been proposed that its positively charged surface can promote the otherwise unstable base pairing between U2 snRNA and the poorly conserved branch point sequence (7, 34). Other mechanisms, including the recruitment of splicing factors involved in prespliceosome formation (5) as well as direct protein-protein interactions with components of U2 snRNP (8), are likely to contribute to U2AF activity.The role of U2AF 35 remains controversial. In vivo analyses of Drosophila U2AF have shown that both subunits, as well as the interaction between them, are essential for viability (14,22,23). In contrast, biochemical complementation experiments performed with extracts chromatographically depleted of U2AF have indicated that U2AF 65 alone is able to provide U2AF activity when tested with model splicing substrates (40,41). Similar results were obtained with nuclear extracts immunodepleted with a monoclonal antibody against U2AF 65 (6, 13). Results obtained with a U2AF 65 mutant protein deficient in its interaction with U2AF 35 also indicated that U2AF 35 is dispensable for in vitro splicing of various pre-mRNAs, including substrates whose splicing depends on the presence of exonic splicing enhancers (13). These sequences are often found downstream of weak 3Ј splice sites (31, 37) and stimulate early events in spliceosome assembly (16, 28), including U2AF 65 binding (36,43).In contrast with these results, immunodepletion with antibodies against U2AF35 resulted in nuclear extracts that required addition of both U2AF 65 and U2AF 35 for efficient splicing of constitutive and exon enhancer-dependent substrates (43). Also supporting an essential role for U2AF 35 were results obtained with a U2AF 65 mutant defective in interaction with U2AF 35 , which was inactive in those assays (43). Based on results obtained in a reconstituted system using limiting amounts of recombinant proteins, and also based on previous data reporting protein-protein interac...

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

“…It is likely that stronger BP 0 (in terms of U2 snRNA complementarity) can compensate for lower AG affinity to U2AF, leading to the recognition of BP 0 in a U2AF-independent manner (or less dependent than in the case of canonical BP). This model is supported by our U2AF depletion experiments and is consistent with previous findings that BP recognition may depend or not on AG binding to U2AF35, according to BP and 3 0 ss sequence and organization 11,[16][17][18] . In contrast, SF3B1 WT may allow a more promiscuous binding of U2 snRNA to both canonical and alternative BPs, and in this case, the final choice of BP may be determined by context, especially 3 0 ss affinity for U2AF.…”

“…15). The low frequency of G nucleotide immediately following alternative AG 0 sites suggests lesser dependence to U2AF1 compared with the canonical AG at the step of 3'ss recognition [16][17][18] . To test this hypothesis, MP41 and HEK293T cells were transiently transfected with siU2AF1 or siU2AF2.…”

Dramatically reduced spliceosome in Cyanidioschyzon merolae

“…This observation may explain its absence in C. merolae, in which this distance averages 29 nt. In contrast, human Mud2 (U2AF2), which binds the polypyrimidine tract, was found to be critical for excision of all introns tested (57). Because there is no polypyrimidine tract in C. merolae (6), we suggest that CmMud2 could bind the branch site directly, as has been reported in S. cerevisiae (58), which also has less prominent polypyrimidine tracts.…”

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

“…In support of this model, several studies demonstrated increased U2AF LG binding to 3Ј-splice sites in an ESE-dependent manner in nuclear extracts (Wang et al 1995;Zuo and Maniatis 1996;Graveley et al 2001). However, other studies failed to find increased ESE-dependent U2AF cross-linking (Guth et al 1999;Kan and Green 1999;Li and Blencowe 1999), possibly due to different experimental conditions (Guth et al 2001). In one instance, an ESE promoted splicing by counteracting the negative effect of a downstream splicing silencer (Kan and Green 1999;Shen et al 2004a).…”