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.     

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.     

Human transformer 2beta and SRp55 interact with a calcitonin-specific splice enhancer

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. Sequences in the human calcitonin-specific fourth exon function as an exonic splice enhancer (ESE) which is required for incorporation of exon 4 into calcitonin mRNA. Deletion of these sequences from the rat calcitonin/CGRP gene was reported to have no effect on calcitonin splicing. We demonstrate that sequences in the rat calcitonin/CGRP fourth exon act as an ESE. In addition, we observed that three proteins in HeLa nuclear extract, of apparent molecular weights of 40, 55 and 85 kDa, specifically interact with the exon 4 ESE. The 40-kDa protein is human transformer 2beta (hTra2beta), a homolog of the Drosophila splice regulator transformer 2. hTra2beta is required for calcitonin splicing in vitro, one of the first biological functions identified for hTra2beta. The 55-kDa protein is SRp55, a member of the SR family of phosphoproteins. Binding of SRp55 to an ESE required for calcitonin mRNA splicing suggests that the different levels of SRp55 present in different cell types may regulate calcitonin/CGRP alternative splicing.     

剪接因子U2AF65相关蛋白的研究进展

U2核糖核蛋白小体辅助因子(U2AF)65是参与前体mRNA剪接的重要辅助因子,前体RNA生成之初,U1核糖核蛋白小体(snRNP)结合到内含子的5'剪接位点,U2AF65和U2AF35分别结合到多聚嘧啶序列和3'剪接位点,剪接因子1(SF1)结合到分支位点是剪接体形成的第一步。U2AF的存在又辅助U2snRNP代替SF1结合到分支位点,使剪接反应顺利进行。最近几年,发现基因组中存在一些U2AF65的旁系同源基因序列。这些旁系同源基因由祖先基因经连续复制而横向形成,复制出的基因副本经历了各自的进化途径,最终它们在结构和功能上有相似之处,又各有独特之处。我们简要讨论了U2AF65、PUF60、CAPERα和CAPERβ这4种同源蛋白的发现过程、结构特征、自身的多样性、基因的进化和生物学功能。     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.     

Alternative modes of binding by U2AF65 at the polypyrimidine tract

During initial recognition of an intron in pre-mRNA, the 3' end of the intron is bound by essential splicing factors. Notably, the consensus RNA sequences bound by these proteins are highly degenerate in humans. This raises the question of 3' splicing factor function in introns lacking canonical binding sites. Investigating the introns of the model organism Neurospora crassa revealed a different organization at the 3' end of the intron compared to most eukaryotic organisms. The predicted branch point sequences of Neurospora introns are much closer to the 3' splice site compared to those in human introns. In addition, Neurospora introns lack the canonical polypyrimidine tract found at the end of introns in most eukaryotic organisms. The large subunit of the U2 snRNP associated factor (U2AF65), which is essential for splicing of human introns and specifically recognizes the polypyrimidine tract, is also present in Neurospora. We show that Neurospora U2AF65 binds RNA with low affinity and specificity, apparently evolving with its disappearing binding site. The arginine/serine rich domain at the N-terminus of Neurospora U2AF65 regulates its RNA binding. We find that this regulated binding can be recapitulated in human U2AF65 which has been mutated to decrease both affinity and overall charge. Finally, we show that the addition of the small U2AF subunit (U2AF35) to U2AF65 with weakened RNA binding affinity significantly enhances the affinity of the resulting U2AF heterodimer.     

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.     

Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF(65) were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.     

Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals.

The first AG dinucleotide downstream from the branchpoint sequence (BPS) is chosen as the 3'' splice site during catalytic step II of the splicing reaction. The mechanism and factors involved in selection of this AG are not known. Early in mammalian spliceosome assembly, U2AF65 binds to the pyrimidine tract between the BPS and AG. Here we show that U2AF65 crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evidence that all three proteins are components of U5 snRNP. These proteins interact with pre-mRNA in the region spanning from immediately downstream of U2 snRNP''s binding site at the BPS to just beyond the 3'' splice site. We also demonstrate that there are strict constraints on both the sequence and the distance between the BPS and AG for catalytic step II. Together, these observations suggest that U5 snRNP is positioned on the 3'' splice site by an interaction (direct or indirect) with U2 snRNP bound at the BPS and by a direct interaction with the pyrimidine tract. The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.     

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.     

The 5'' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.     

Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.     

The SF3b155 N-terminal domain is a scaffold important for splicing

Recruitment of U2 snRNP to the branch point sequence of introns is a necessary step in pre-mRNA splicing. In the current model, U2AF65, bound at the polypyrimidine tract of the intron, recruits U2 snRNP to the branch point sequence by interacting with the U2 snRNP protein SF3b155. We demonstrate that the N-terminal domain of SF3b155 contains multiple U2AF65 binding sites that are distinct from the binding site for the U2 snRNP protein p14, mapped to amino acids 396-424 of SF3b155. The N-terminal domain of SF3b155 appears to adopt a primarily unfolded structure but is functional to inhibit splicing in vitro. RNA binding studies show that the N-terminal domain of SF3b155 binds RNA nonspecifically and that the sites for U2AF65 binding and RNA binding are overlapping (or the same) within SF3b155. We propose that the N-terminal domain of SF3b155 adopts a primarily unfolded structure that functions as a scaffold to facilitate SF3b155's multiple protein-protein and protein-RNA interactions. The multiple U2AF65 binding sites on SF3b155 suggest a model in which multiple U2AF65 molecules bound to the intron could enhance U2 snRNP recruitment to the branch point sequence.     

Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF

Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding.     

The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.

The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex-specific alternative splicing of doublesex (dsx) pre-mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre-mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre-mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female-specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site.     

The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit

Splicing of exon 6B from the beta-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.     

A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site

Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.