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.     

PUF60: a novel U2AF65-related splicing activity

We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.     

Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65

The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.     

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.     

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.     

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal

The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine > adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.     

Fas splicing regulation during early apoptosis is linked to caspase-mediated cleavage of U2AF65

U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) is an essential splicing factor in the recognition of the pre-mRNA 3' splice sites during the assembly of the splicing commitment complex. We report here that U2AF65 is proteolyzed during apoptosis. This cleavage is group I or III caspase dependent in a noncanonical single site localized around the aspartic acid(128) residue and leads to the separation of the N- and C-terminal parts of U2AF65. The U2AF65 N-terminal fragment mainly accumulates in the nucleus within nuclear bodies (nucleoli-like pattern) and to a much lesser extent in the cytoplasm, whereas the C-terminal fragment is found in the cytoplasm, even in localization studies on apoptosis induction. From a functional viewpoint, the N-terminal fragment promotes Fas exon 6 skipping from a reporter minigene, by acting as a dominant-negative version of U2AF65, whereas the C-terminal fragment has no significant effect. The dominant-negative behavior of the U2AF65 N-terminal fragment can be reverted by U2AF35 overexpression. Interestingly, U2AF65 proteolysis in Jurkat cells on induction of early apoptosis correlates with the down-regulation of endogenous Fas exon 6 inclusion. Thus, these results support a functional link among apoptosis induction, U2AF65 cleavage, and the regulation of Fas alternative splicing.     

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.     

Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.     

A conditional role of U2AF in splicing of introns with unconventional polypyrimidine tracts

Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3' splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3' splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3' splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.     

Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.     

Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60

PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.     

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro

The general splicing factor U2AF(65) recognizes the polypyrimidine tract (Py tract) that precedes 3' splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF(65) activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF(65) in vitro. However, deletion of the RRM3 domain of S. pombe U2AF(59) abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded beta-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.     

Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65)

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.     

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.     

Genomic mRNA profiling reveals compensatory mechanisms for the requirement of the essential splicing factor U2AF

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3' splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF(59) (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF(59) inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF(59)-insensitive introns have stronger 5' splice sites and higher A/U content. Most importantly, features that contribute to U2AF(59) insensitivity of an intron unexpectedly reside in its 5'-most 30 nucleotides. These include the 5' splice site, a guanosine at position 7, and the 5' splice site-to-branch point sequence context. A differential requirement (similar to U2AF(59)) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.     

Identification of motifs that function in the splicing of non-canonical introns

Background  

While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.