Interaction between the negative regulator of splicing element and a 3' splice site: requirement for U1 small nuclear ribonucleoprotein and the 3' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.     

An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.     

Fas (APO-1/CD95)-mediated apoptosis is independent of bcl-2: a study with cell lines overexpressing bcl-2 and with bcl-2 transfected cell lines

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.     

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.     

Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing

We have identified a class of pre-mRNAs that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, we described pre-mRNAs that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon.     

A coactivator of pre-mRNA splicing

The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.     

The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites

Efficient splicing of the 5'-most intron of pre-mRNA requires a 5' m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP binding to 5' splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5' cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5' splice site and not with any loss of U1 snRNP binding.     

Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5' splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5' splice site and by an exonic enhancer differ mechanistically.     

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.     

Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA

This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.     

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.     

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.     

The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.     

RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50DeltaRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP?

Psoralen cross-linking experiments in HeLa cell nuclear extracts have revealed the binding of U1 snRNA to substrates containing the SV40 late and adenovirus L3 polyadenylation signals. The sites of U1 cross-linking to the substrates map different distances upstream of the AAUAAA sequence to regions with limited complementarity to the 5' end of U1 snRNA. U1 cross-linking to the same site in the SV40 late pre-mRNA is enhanced by the addition of an upstream 3' splice site, which also enhances polyadenylation. Examination of different nuclear extracts reveals a correlation between U1 cross-linking and the coupling of splicing and polyadenylation, suggesting that the U1 snRNP participates in the coordination of these two RNA-processing events. Mutational analyses demonstrate that U1/substrate association cannot be too strong for coupling to occur and suggest that the U1 snRNP plays a similar role in recognition of internal and 3' terminal exons. Possible mechanisms for communication between the splicing and polyadenylation machineries are discussed, as well as how interaction of the U1 snRNP with 3' terminal exons might contribute to mRNA export.     

The role of U5 snRNP in pre-mRNA splicing

The current model for the function of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome proposes that U5 carries binding sites for the 5' and 3' exons, allowing the spliceosome to 'tether' the 5' exon intermediate produced by the first catalytic step and align it with the 3' exon for the second step. Functional analysis of U5 snRNA in cis-spliceosomes has provided support for this model, and data from nematode and trypanosome splicing systems suggest that U5 or a U5-like snRNA performs a similar role in trans-splicing.     

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.