A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus.

Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the SR [corrected] family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55). In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated cross-linked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55. The possible roles of hnRNP H in NRS function are discussed.

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.

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element.

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated 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.

SR protein and snRNP requirements for assembly of the Rous sarcoma virus negative regulator of splicing complex in vitro.

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.

Characterization of an RNP complex that assembles on the Rous sarcoma virus negative regulator of splicing element.

We have characterized an RNP complex that assembles in nuclear extracts on the negative regulator of splicing (NRS) element from Rous sarcoma virus. While no complex was detected by native gel electrophoresis under conditions that supported spliceosome assembly, gel filtration revealed a specific ATP-independent complex that rapidly assembled on NRS RNA. No complexes were formed on non-specific RNA. Unlike the non-specific H complex, factors required for NRS complex assembly are limiting in nuclear extract. The NRS complex was not detected in reactions containing ATP and pre-formed complexes were dissociated in the presence of ATP. In addition, the assembly process was sensitive to high salt but NRS complexes were salt stable once formed. Assembly of the NRS complex appears functionally significant since mutated NRS RNAs that fail to inhibit splicing in vivo are defective for NRS complex assembly in nuclear extract. The probable relationship of the NRS complex to spliceosomal complexes is discussed.

SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element.

Retroviral replication requires that a portion of the primary transcripts generated from proviral DNA be spliced to serve as mRNA for the envelope protein and in Rous sarcoma virus as src mRNA. However, a substantial amount of full-length RNA must be maintained in an unspliced form, as the unspliced RNA serves both as mRNA for structural proteins and virion-associated enzymatic proteins and as genomic RNA for progeny virions. The extent of viral RNA splicing must be finely controlled, since only a narrow range in the ratio of unspliced RNA to spliced RNA is tolerated for optimal replication. A number of cis-acting sequences within the RNA of Rous sarcoma virus play a role in preserving a large pool of unspliced RNA. One such sequence, the negative regulator of splicing (NRS), is of interest because it blocks splicing but is not located near any of the splice junctions. To better understand how this novel element blocks splicing at a distance, we set out to identify host cell factors that interact specifically with this inhibitory sequence. In this study, proteins from nuclear extracts with molecular masses of 26, 36, 44, and 55 kDa were shown by UV cross-linking assays to bind the NRS preferentially. One of them, p55, was also detected in a specific complex with SR protein electrophoretic mobility shift assay. All but p55 have biochemical properties consistent with SR protein splicing factors, and some, but not all, of the total SR proteins purified from HeLa cells cross-link specifically to the NRS. The strongest cross-linking SR protein is SRp30a/b, which is composed of the splicing factors SF2/ASF and SC35. The NRS specifically binds bacterially expressed SF2/ASF, whereas nonfunctional mutants do not. Data indicating that the 36-kDa protein which cross-links in nuclear extracts is SF2/ASF are presented. The data indicate that factors normally required for RNA splicing may be exploited by retroviruses to block splicing.

Two strong 5' splice sites and competing, suboptimal 3' splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA.

The human immunodeficiency virus type 1 (HIV-1) genome contains 20 exons that are alternatively spliced from 16 splice sites to generate more than 40 different mRNAs, including incompletely spliced and unspliced mRNAs. In contrast to avian retroviral RNA, which has a cis-acting element in gag that negatively regulates splicing (NRS), HIV-1 RNA did not have any NRS sequences in the gag or pol genes detectable by a splicing inhibition assay. However, this assay demonstrated that the HIV-1 first 5' splice site competed with a cellular 5' splice site, suggesting that HIV-1 may have some strong splice sites. To extend this observation, we used a splice site swapping strategy to determine the efficiency of 14 HIV-1 splice sites in human beta globin chimeras tested in transient transfection experiments. While the 1st HIV-1 5' splice site used in all spliced transcripts and the 4th 5' splice site used in most of the 2-kb transcripts were efficient, the other splice sites, including all the 3' splice sites, were less efficient, ranging in use from 25 to 60%. We propose that this range of splice site efficiencies contributes to the regulation of alternative splicing of HIV-1 mRNAs.

Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing.

A cis-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U11 and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U11 and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U11 and U12 snRNPs in the inhibition of splicing mediated by the RSV NRS RNA.

Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.

Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing.

Retroviruses splice only a fraction of their primary RNA transcripts to subgenomic mRNA. The unspliced RNA is transported to the cytoplasm, where it serves as genomic RNA as well as mRNA for the gag and pol genes. Deletion of sequences from the Rous sarcoma virus gag gene, which is part of the intron of the subgenomic mRNAs, was previously observed to result in an increase in the ratio of spliced to unspliced RNA. These sequences, which we termed a negative regulator of splicing (NRS), can be moved to the intron of a heterologous gene resulting in an accumulation of unspliced RNA in the nucleus. We have used such constructs, assayed by transient expression in chicken embryo fibroblasts, to define the minimal sequences necessary to inhibit splicing. Maximal NRS activity was observed with a 300-nt fragment containing RSV nts 707-1006; two noncontiguous domains within this fragment, one of which contains a polypyrimidine tract, were both found to be essential. The NRS element was active exclusively in the sense orientation in two heterologous introns tested and in both avian and mammalian cells. Position dependence was also observed, with highest activity when the NRS was inserted in the intron near the 5' splice site. The NRS element was also active at an exon position 136 nts upstream of the 5' splice site but not at sites further upstream. In addition, it did not affect the splicing of a downstream intron.

Deoxyglucose-resistant mutants of Neurospora crassa: isolation, mapping, and biochemical characterization.

Neurospora crassa mutants resistant to 2-deoxyglucose have been isolated, and their mutations have been mapped to four genetic loci. The mutants have the following characteristics: (i) they are resistant to sorbose as well as to 2-deoxyglucose; (ii) they are partially or completely constitutive for glucose transport system II, glucamylase, and invertase, which are usually repressed during growth on glucose; and (iii) they synthesize an invertase with abnormal thermostability and immunological properties, suggesting altered posttranslational modification. All of these characteristics could arise from defects in the regulation of carbon metabolism. In addition, mutants with mutations at three of the loci lack glucose transport system I, which is normally synthesized constitutively by wild-type N. crassa. Although the basis for this change is not yet clear, the mutants provide a way of studying the high-affinity system II uncomplicated by the presence of the low-affinity system I.

Isolation and characterization of a Neurospora glucose-repressible gene.

Using differential hybridization, the cDNA copy of a Neurospora gene coding for an abundant glucose-repressible mRNA (grg-1) has been isolated. The cDNA was used to clone the genomic copy, and both were sequenced. The cDNA is nearly full length and contains putative translational start and termination codons. Conceptual translation indicates that grg-1 mRNA could direct the synthesis of a 7,000 molecular weight polypeptide. The genomic clone, contained in an 1,888 bp PvuII fragment, encompasses the entire cDNA as well as 838 bp of 5' and 369 bp of 3' flanking sequence. Comparison of the cDNA and genomic clones revealed the presence of two short introns in potential protein-coding sequences. grg-1 message levels were found to increase within minutes following the onset of glucose deprivation and rise 50 fold during the first 90 min of derepression.

Neurospora glucamylase and a mutant affected in its regulation.

Neurospora glucamylase is a glucose-repressible extracellular enzyme. The enzyme was purified to homogeneity and found to have a molecular weight of 82,000 and to release glucose from either maltose or amylose. The rate of glucamylase synthesis increases more than 100-fold when cells are transferred from a glucose-containing medium to a glucose-free medium. Increased from a glucose-containing medium to a glucose-free medium. Increased production of glucamylase begins within 30 min of the transfer. Glucamylase is rapidly secreted into the medium. A mutant affecting the ability of glucose to repress the synthesis of the glucose-repressible extracellular enzymes glucamylase and invertase has been isolated and studied. The mutant constitutively synthesizes and secretes a glucamylase which is indistinguishable from the wild-type enzyme.