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.     

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.     

Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron

Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.     

Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

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.     

The U1, U2 and U5 snRNAs crosslink to the 5' exon during yeast pre-mRNA splicing

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing.     

Polypyrimidine tract binding protein blocks the 5' splice site-dependent assembly of U2AF and the prespliceosomal E complex

Polypyrimidine tract binding protein (PTB) represses some alternatively spliced exons by direct occlusion of splice sites. In repressing the splicing of the c-src N1 exon, we find that PTB acts by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5' splice site. Instead, PTB prevents formation of the prespliceosomal early (E) complex across the intervening intron by preventing the assembly of the splicing factor U2AF on the 3' splice site of exon 4. When the unregulated 5' splice site of the upstream exon 3 is present, U2AF binding is restored and splicing between exons 3 and 4 proceeds in spite of the N1 exon bound PTB. Thus, rather than directly blocking the N1 splice sites, PTB prevents the 5' splice site-dependent assembly of U2AF into the E complex. This mechanism likely occurs in many other alternative exons.     

Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

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Highlights? Splicing factor SF1 phosphorylation on a conserved SPSP motif is required in vivo ? SPSP phosphorylation (P) induces local folding within an SF1/U2AF⁶⁵ interface ? Phosphorylation promotes an acutely bent (P)SF1/U2AF⁶⁵/RNA conformation     

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.     

Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL,the U1 snRNP and U2AF underlie male exon skipping

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.     

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.     

Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins which stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. We have tested this model by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) bound equally to the pre-mRNA in the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa bound prior to most SR proteins and hTra2beta and its level of binding did not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA was, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract.     

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.     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.