Characterization of U2AF6, a Splicing Factor Related to U2AF35

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.     

The role of U2AF35 and U2AF65 in enhancer-dependent splicing.

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.     

Plant-specific SR-related protein atSR45a interacts with spliceosomal proteins in plant nucleus

Serine/arginine-rich (SR) protein and its homologues (SR-related proteins) are important regulators of constitutive and/or alternative splicing and other aspects of mRNA metabolism. To clarify the contribution of a plant-specific and stress-responsive SR-related protein, atSR45a, to splicing events, here we analyzed the interaction of atSR45a with the other splicing factors by conducting a yeast two-hybrid assay and a bimolecular fluorescence complementation analysis. The atSR45a-1a and -2 proteins, the presumed mature forms produced by alternative splicing of atSR45a, interacted with U1-70K and U2AF³⁵b, splicing factors for the initial definition of 5′ and 3′ splice sites, respectively, in the early stage of spliceosome assembly. Both proteins also interacted with themselves, other SR proteins (atSR45 and atSCL28), and PRP38-like protein, a homologue of the splicing factor essential for cleavage of the 5′ splice site. The mapping of deletion mutants of atSR45a proteins revealed that the C-terminal arginine/serine-rich (RS) domain of atSR45a proteins are required for the interaction with U1-70K, U2AF³⁵b, atSR45, atSCL28, PRP38-like protein, and themselves, and the N-terminal RS domain enhances the interaction efficiency. Interestingly, the distinctive N-terminal extension in atSR45a-1a protein, but not atSR45a-2 protein, inhibited the interaction with these splicing factors. These findings suggest that the atSR45a proteins help to form the bridge between 5′ and 3′ splice sites in the spliceosome assembly and the efficiency of spliceosome formation is affected by the expression ratio of atSR45a-1a and atSR45a-2.     

RS domain-splicing signal interactions in splicing of U12-type and U2-type introns

Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine/serine-rich (RS) domain. On typical U2-type introns, RS domains contact the branchpoint and 5' splice site to promote base-pairing with U small nuclear RNAs (snRNAs). Here we analyze the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We show that RS domains contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, we find that the RS domain contacts the site of the U6 snRNA-5' splice site interaction during the first step of splicing and shifts to contact the site of the U5 snRNA-exon 1 interaction during the second step. Our results reveal alternative interactions between the RS domain and 5' splice site region that coincide with remodeling of the spliceosome between the two catalytic steps.     

Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF

The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3' splice site that serves as an important signal for both constitutive and regulated pre-mRNA splicing. The small subunit U2AF(35) interacts with the 3' splice site dinucleotide AG and is essential for regulated splicing. Like several other proteins involved in constitutive and regulated splicing, both U2AF(65) and U2AF(35) contain an arginine/serine-rich (RS) domain. In the present study we determined the role of RS domains in the subcellular localization of U2AF. Both U2AF(65) and U2AF(35) are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF(65) or U2AF(35) acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this factor to initiate spliceosome assembly and therefore contribute to regulate splicing.     

U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans

In most species the 3' splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3' splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.     

Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing

Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.     

A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly

Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF(65) and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5' splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.     

The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase.

SR proteins play important roles in the recognition and selection of the 3' and 5' splice site of a given intron and contribute to the phosphorylation/dephosphorylation-mediated regulation of pre-mRNA splicing. Recent studies have demonstrated that the U1 snRNP is recruited to the 5' splice site by protein/protein interactions involving the SR domains of the U1-70K protein and SF2/ASF. Recently, it was suggested that SR proteins might also contribute to the binding of the [U4/U6.U5] tri-snRNP to the pre-spliceosome (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706), although it remains unclear whether these SR proteins interact with proteins of the tri-snRNP complex. As a first step toward the identification of proteins that could potentially mediate the integration of the [U4/U6.U5] tri-snRNP complex into the spliceosome, we investigated whether purified [U4/U6.U5] tri-snRNP complexes contain SR proteins. Three proteins in the tri-snRNP complex with approximate molecular weights of 27, 60, and 100 kDa were phosphorylated by purified snRNP-associated protein kinase, which has been shown previously to phosphorylate the serine/ arginine-rich domains of U1-70K and SF2/ASF (Woppmann A et al., 1993, Nucleic Acids Res 21:2815-2822). These proteins are thus prime candidates for novel tri-snRNP SR proteins. Here, we describe the biochemical and molecular characterization of the 27K protein. Analysis of a cDNA encoding the 27K protein revealed an N-terminal SR domain strongly homologous (54% identity) to the SR domain of the U1 snRNP-specific 70K protein. In contrast to many other SR proteins, the 27K protein does not contain an RNA-binding domain. The 27K protein can be phosphorylated in vitro by the snRNP-associated protein kinase and exhibits several isoelectric variants upon 2D gel electrophoresis. Thus, the tri-snRNP-specific 27K protein could potentially be involved in SR protein-mediated protein/protein interactions and, additionally, its phosphorylation state could modulate pre-mRNA splicing.     

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex.

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.