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PubMed1993

A shortened form of the Tetrahymena thermophila group I intron can catalyze the complete splicing reaction in trans.

The group I intron from Tetrahymena thermophila is able to catalyze its own excision from a precursor RNA. The intron recognizes the splice sites through an intron-encoded sequence called the internal guide sequence, or IGS. The 5' and 3' exons are thought to align on the IGS and form a pseudoknot structure consisting of two stems (P1 and P10). We created a shortened form of the intron that lacks the exon sequences and the entire IGS. This RNA is unable to react upon itself. It can catalyze a sequential two-step transesterification reaction on a P1P10 substrate added in trans that completely mimics splicing. The reaction works for different substrates that contain a U.G base-pair preceding the 5' cleavage site and a guanosine base preceding the 3' cleavage site, but that are otherwise unrelated in sequence. The ribozyme uses primarily the correct 5' and 3' splice sites even in the presence of potential cryptic splice sites, and therefore it must rely on the structure of the substrate (formation of the P1 and P10 helices) for correct splice site recognition. A C-G base-pair after the 5' splice site in P1 decreases activity while a U.G or G.U base-pair enhances activity. The relative position in P1 of the U.G base-pair preceding the 5' splice site is an important determinant. The ability of the intron to recognize primarily a specific structure, rather than a sequence, has ramifications for splice-site selection, for molecular modeling of the group I intron, and for ribozyme-based gene targeting.

Animals↗
PubMed1994

A chloroplast group I intron undergoes the first step of reverse splicing into host cytoplasmic 5.8 S rRNA. Implications for intron-mediated RNA recombination, intron transposition and 5.8 S rRNA structure.

The chloroplast 23 S rRNA gene of Chlamydomonas reinhardtii contains a self-splicing group I intron, Cr.LSU. Incubation of total RNA from C. reinhardtii with [alpha-32P]GTP produces a GTP-labeled RNA that is derived from Cr.LSU, but is approximately 95 nucleotides longer (and referred to as IA). We show that IA is derived from an intermolecular reaction of Cr.LSU with cytoplasmic 5.8 S rRNA; the 3'-terminal G of the intron becomes ligated to A64. This reaction, which was reproduced using synthetic RNAs, is identical to the first step of reverse splicing. The second step of reverse splicing did not occur with any efficiency, even though both P1 and P10-like helices can form between the reactive region of 5.8 S rRNA and the IGS of Cr.LSU. Cloning of IA provided a chimeric Cr.LSU precursor with the normal 3' exon substituted by 95 nucleotides of 5.8 S; this precursor spliced poorly despite having a potential P10 helix (between the 3'-exon and the IGS). The inefficient reverse splicing of Cr.LSU into 5.8 S RNA and poor forward splicing of the chimera may be explained by competition between helices P1 (between the 5'-exon and IGS) and P10; competition between P1 and P10 is apparently not a factor in forward splicing of the wild-type LSU precursor. The sequence of the 5.8 S gene of C. reinhardtii was determined, and the published RNA sequence revised. A secondary structure model of C. reinhardtii 5.8 S rRNA was proposed incorporating the requirement for base-pairing to the IGS of Cr.LSU. The resulting structure resembles that recently proposed for yeast 5.8 S rRNA, and has considerable advantages over previous models of C. reinhardtii 5.8 S rRNA. These data describe a novel example of a naturally occurring ribozyme reacting with a naturally occurring RNA of the same cell; implications for ribozyme-mediated RNA recombination and intron transposition via reverse splicing are discussed.

Animals↗
PubMed1996

Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products.

Self-splicing group II introns are found in bacteria and in the organellar genes in plants, fungi, and yeast. The mechanism for the first step of splicing is generally believed to involve attack of a specific intronic 2'-hydroxyl group on a phosphodiester linkage at the 5'-splice site, resulting in the formation of a lariat intron species. In this paper, we present kinetic and enzymatic evidence that in vitro there are two distinct pathways for group II intron self-splicing: one involves 2'-OH attack and another involves attack of water or hydroxide. These two pathways occur in parallel under all reaction conditions, although either can dominate in the presence of particular salts or protein cofactors. Both pathways are followed by a successful second step of splicing, and either pathway can be highly efficient. We find that the hydrolytic pathway prevails under physiological ionic conditions, while branching predominates at molar concentrations of ammonium ion. The intron is observed to adopt two major active conformations. In order to quantify their individual reaction rates, we applied a mechanistic model describing biphasic parallel kinetic behavior. Kinetic analysis throughout the investigation reveals that there is no coupling between the unproductive "spliced-exon-reopening" reaction (SER) and hydrolysis during the first step of splicing. Conditions that stimulate branching can promote the SER reaction just as efficiently as conditions that stimulate the hydrolytic pathway. Although there is little evidence that it exists in vivo, a hydrolytic splicing pathway for group II introns has important implications for the translation of intron-encoded proteins and the inhibition of intron migration into new genomic positions.

Ammonium Chloride↗
PubMed2001

Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.

Adenosine Monophosphate↗
PubMed1997

Automated electron tomography of large nuclear RNP (InRNP) particles--the naturally assembled complexes of precursor messenger RNA and splicing factors.

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to the cytoplasm to function in protein synthesis. Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA, are packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200S region in sucrose gradients, contain all U snRNPs required for pre-mRNA splicing and several protein splicing factors, including U2AF and the SR proteins and can thus be viewed as naturally assembled complexes of pre-mRNA and splicing factors. We have previously reconstructed the three-dimensional image of negatively stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, composed of four major subunits. Here we further analyzed the reconstructed models and the apparent connectivity between the subunits using a new rendering technique. The uniformity of the lnRNP particles was substantiated by measurement of the volume engulfed by their surface. This study further supports the model proposed for the packaging of nuclear pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner.

Animals↗
PubMed2000

Expression of neurexin Ialpha splice variants in sympathetic neurons: selective changes during differentiation and in response to neurotrophins.

Neurexins are a surprisingly diverse group of alternatively spliced proteins possibly involved in neural cell recognition processes. We find neurexin Ialpha and its splice variants highly conserved between mammals and birds. In vivo, neurexin Ialpha is expressed in sympathetic neurons during target innervation and relative expression levels of splice variants change with development. In vitro, no such changes are observed in the absence of growth factors, indicating that interactions with the environment are required to modify the splicing pattern. Specific alterations in splice variant expression are induced in vitro by neurotrophins. Expression patterns of splice variants in vivo and neurotrophin-induced regulation without changes in cell composition in vitro demonstrate that neurexin splice variant expression varies during differentiation of individual neurons. Our data suggest that changes in neurexin splice variants contribute to alterations of neuronal cell surface properties during target innervation.

Alternative Splicing↗
PubMed1994

Fine-mapping of sequences that suppress splicing in the E3 complex transcription unit of adenovirus.

Complex transcription units encode multiple mRNAs that arise by alternative processing of a common pre-mRNA. Some mechanism must exist that maintains the balance among the various mRNAs. The E3 complex transcription unit of adenovirus encodes four major mRNAs (termed a, c, f, and h) and several minor mRNAs (d, e, i). mRNAs a and c account for about one-half of E3 mRNAs. Using virus deletion mutants we have identified a region (termed Region I) which, when deleted, leads to the formation of doubly spliced mRNAs f and h at the expense of singly spliced mRNAs a and c. Region I consists of at most 129 nucleotides. Deletions as small as 24 nucleotides markedly abolished the splice-suppressing function of Region I. We propose that Region I normally functions to suppress splicing in the E3 pre-mRNA and thereby allow mRNA a and c to be formed in abundance. We have also identified another region, designated as Region IA, that appears to specifically suppress splicing to form the doubly-spliced minor mRNAs d and e. Neither Region I nor Region IA include a conventional splice site or cleavage/polyadenylation site. We speculate that Region I and Region IA probably suppress splicing by binding a trans-acting factor.

Adenoviridae↗
PubMed1997

Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA.

Defective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional "spliced" genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/ core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis.

Cloning, Molecular↗
PubMed1995

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts.

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans spliced introns.

Animals↗
PubMed1994

In vitro splicing of pre-mRNA containing bromouridine.

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1,2]. We have investigated the effect of bromouridine (BrU) in pre-mRNA on the efficiency of splicing. An adenovirus major late II construct was used to prepare four different transcripts, each containing a different amount of BrU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in the transcript were substituted for BrU. Splicing was restored to some extent if 50% of the Us were replaced by BrU. The splicing efficiency returned to an almost normal level if only 1 our of every 10 Us was substituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally in vitro. Furthermore, these results strongly suggest that some Us in the adenoviral transcript, probably those at the splice sites, cannot be replaced by BrU and are therefore critical in the splicing reaction.

Adenoviridae↗
PubMed2003

Amelogenin gene splice products: potential signaling molecules.

The amelogenins, the major proteins of the developing tooth enamel matrix, are highly conserved throughout most species studied. The gene structure is similar, with a set of seven exons and intervening introns, and remarkable conservation of particular exon sizes over divergent species. Studies of exon skipping and consequent alternative gene splicing suggest that, in vertebrates, exon definition is crucial. In this mechanism, exon size is important. If too small, an exon can be readily skipped, if too large, internal cryptic splice sites may be utilized. Other factors, such as intron length and specific nucleotide sequences at the splice boundaries also modulate splicing efficiency, but amelogenin gene splicing conforms well to the generalized exon length model. Exons 1, 2 and 7 are not subject to splicing that affects the secreted protein product, but exons 3, 4 and 5 are at the lower boundary of exon size, rendering them, 4 and 5 especially, subject to skipping. On the other hand, exon 6 is very long and has cryptic splicing sites that can be used. In the mouse, nine distinct splice product proteins have been detected. The question now is the functions of these products. The larger forms, those that contain the intact proline-rich, hydrophobic exon 6 domains, are important for enamel mineralization. Recent work suggests that the small proteins resulting from deletion of a major part of amelogenin gene exon 6 via utilization of a cryptic site may have signal transduction functions during tooth development. Furthermore, new work also suggests that odontoblasts transiently express the small amelogenins during the period that epithelial-mesenchymal signaling between preodontoblasts and preameloblasts determines the course of tooth development. The same peptides have been demonstrated to act on non-odontogenic cells and effect their phenotypic expression patterns in vitro, and to induce bone formation in implants in vivo.

Alternative Splicing↗
PubMed2005

Identification and expression analysis of alternatively spliced isoforms of human interleukin-23 receptor gene in normal lymphoid cells and selected tumor cells.

Interleukin 23 (IL-23) is a new member of the IL-12 family that plays a critical role in promoting the proliferation of memory T helper 1 cells. The heterodimerized IL-23 receptor is composed of a shared IL-12 receptor beta 1 (IL-12Rbeta1) and an IL-12Rbeta2-related molecule called IL-23R. The standard form of IL-23R is encoded by at least 12 exons. Here, we demonstrate that at least six spliced isoforms of IL-23R (IL-23R1 to 6) can be generated through alternative splicing. The splicing strategies for the IL-23R gene are complicated and most often result in the deletion of exon 7 and/or exon 10. Translation prediction revealed that these spliced variants result in either premature termination to give rise to a diverse form of receptor ectodomain, or a frameshift to generate various lengths of the IL-23R endodomain. Differential expressions of IL-23R spliced variants are observed in natural killer and CD3+ CD4+ T cells. The expressions of these spliced variants are also prevalently and complicatedly regulated in tumor cell lines. Interestingly, only IL-23R2 and/or IL-23R4 variants are predominantly detected in certain human lung carcinomas, but not in their resected normal margin tissues. Thus, our results indicate that the regulation of alternative splicing on the IL-23R gene is complicated, and the preferential expression of certain IL-23R spliced variants may be a contributive factor to the pathogenesis of certain cancers.

Alternative Splicing↗
PubMed2026

Bioinformatic analysis reveals the potential association of ESRP1 with the splicing of cytoskeleton-associated genes in doxorubicin-resistant MCF7 breast cancer cells.

BACKGROUND: Breast cancer remains one of the most prevalent malignancies among women, with doxorubicin resistance posing a significant challenge that undermines treatment success and survival outcomes. Aberrant alternative splicing (AS), driven by dysregulation or mutations in splicing factors (SFs), is implicated in cancer initiation, progression, and drug resistance. This study aims to investigate the association of the epithelial cell-specific splicing factor ESRP1 with doxorubicin resistance in breast cancer, focusing on how ESRP1 deficiency correlates with AS changes that promote chemoresistance. METHODS: We analyzed RNA-sequencing (RNA-seq) data from doxorubicin-resistant (MCF7-DR) and parental (MCF7) breast cancer cell lines to identify enhanced alternative splicing events (ASEs) and changes in ESRP1 expression; we further leveraged The Cancer Genome Atlas (TCGA)-BRCA cohort to construct an SF-RASE correlation network for screening core SFs (including ESRP1). An integrative analysis combining crosslinking immunoprecipitation (CLIP-seq) data and The Cancer Genome Atlas (TCGA) database was performed to validate ESRP1 binding targets and assess the association between ESRP1-related splicing and cytoskeleton organization. RESULTS: We observed extensive AS changes and significantly downregulated ESRP1 expression in MCF7-DR cells. Integrative analysis identified 61 high-confidence ASEs that correlate with ESRP1 expression. Further bioinformatic integration suggests that ESRP1 expression is associated with the splicing patterns of SPTBN1, MAP2K7, FGFR3, and CYB561A3-four genes involved in cytoskeleton organization-though direct experimental verification to confirm a causal regulatory relationship between ESRP1 and the splicing of these genes is still pending. CONCLUSIONS: Our findings suggest that ESRP1 expression is closely associated with doxorubicin resistance in breast cancer cells, with concomitant alterations in key ASEs linked to cytoskeletal remodeling that correlate with ESRP1. Exploring the ESRP1-related splicing network may offer new strategies to overcome chemoresistance and improve patient outcomes. However, the small cell line sample size (n = 2 per group) constrains the robustness of ASE and SF-ASE correlation findings, and these results should be interpreted with caution and require further validation with larger sample cohorts.

Alternative splicing↗
PubMed1991

Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome.

The pX region of human T lymphotropic virus type I (HTLV-I) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X-III. This possibility was examined by amplifying cDNA derived from HTLV-I+ cells between the 5' LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double-spliced pX mRNA as previously reported. The shorter band was derived from a single-spliced mRNA. HTLV-I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X-III seem to be well correlated with a double-spliced and a single-spliced mRNA, respectively.

Base Sequence↗
PubMed1991

Determination of an RNA structure involved in splicing inhibition of a muscle-specific exon.

We have investigated the RNA structure of the region surrounding the muscle-specific exon 6B of the chicken beta-tropomyosin gene. We have used a variety of chemical and enzymatic probes: dimethylsulfate, N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p-tolu enesulfonate) , RNase T1 and RNase V1. Lead acetate was also used to obtain some information on the tertiary structure of this region. Probing the wild-type sequence suggests a model involving one-stem and three-stem-loop structures in and around this exon. Two of these, hairpin I and stem III, have previously been implicated in repression of splicing of the intron following exon 6B in a HeLa nuclear extract. Stem I includes sequences at the beginning of exon 6B and stem III results from interaction of the intron upstream from exon 6B with sequences in the middle of the intron downstream from this exon (the intron whose splicing is repressed). Neither stem I nor stem III directly involves the consensus sequences (5' splice site, branch-point, 3' splice site) of the repressed intron. Probing RNAs that are derepressed for splicing of this intron show that there are structural changes around the 5' splice site and branch-point sequence that correlate with the derepression. This is true, despite the fact that the derepressed RNAs are altered in a region far from these consensus sequences. The most striking structural correlation with splicing capacity of the intron downstream from exon 6B is seen by probing with lead acetate. Lead ions cut RNA at specific residues; these sites are very sensitive to RNA tertiary structure. Repressed and derepressed RNAs show entirely different cleavage patterns after incubation with lead acetate. Remarkably, hybridizing a derepressed RNA with an RNA comprising the ascending arm of stem III not only re-establishes repression, but also converts the pattern of susceptibility to attack by lead ions over the whole molecule. We suggest that RNA conformation plays a role in keeping exon 6B from being spliced into non-muscle cell mRNA.

Animals↗
PubMed1991

Analysis of spliced and unspliced Rous sarcoma virus RNAs early and late after infection of chicken embryo fibroblasts: effect of cell culture conditions.

Quantitative determinations of spliced and unspliced viral RNA early after retrovirus infection or after transient transfection with proviral DNA have been difficult because of low intracellular viral RNA levels. In this report we describe conditions for the sensitive and quantitative assay of unspliced viral RNA and spliced mRNAs from avian sarcoma virus-infected and transiently transfected chicken embryo fibroblasts. RNase protection mapping was performed with a tandem 32P-labeled riboprobe consisting of three separate regions complementary to the major 5' splice site and the two major 3' splice sites (env and src). The steady-state levels of viral RNA species were determined as early as 14 hr postinfection; no significant changes were observed in the relative levels of spliced mRNAs or in splice site usage as the infection progressed from 14 to 48 hr. After the cells became morphologically transformed at approximately 72 hr postinfection, the condition of the medium of the transformed cell culture affected the steady-state levels of viral RNAs. Unspliced genomic RNA predominated in the transformed cells at 4.5 to 8 hr after feeding; spliced env mRNA was the major species of RNA at 46 to 103 hr after medium change. In contrast, the steady-state levels of viral RNA in transiently transfected cells or in cells infected with a transformation-defective avian sarcoma virus were not as sensitive to the conditions of medium change. The medium-dependent changes in steady-state levels of viral RNA species may either be caused by a declining transcription rate resulting in the accumulation of the more stable env mRNA relative to the more labile unspliced RNA and src mRNA or to changes in the extent of viral RNA splicing.

Animals↗
PubMed1991

Simian immunodeficiency virus (SIVmac) exhibits complex splicing for tat, rev, and env mRNA.

The simian immunodeficiency virus (SIV) is a T-lymphotropic lentivirus associated with a fatal AIDS-like disease in rhesus macaques. SIV has a complex genome encoding virion structural proteins, transactivators, and accessory genes. From lymphoid cells chronically infected with a biologically active molecular clone of SIV, SIVmac1A11, the polymerase chain reaction technique has been used to selectively amplify transcripts for viral transactivators and the envelope gene. Three species of mRNA encoding only rev, and three mRNA encoding both rev and tat were identified by nucleotide sequence analysis. They differed in the splice acceptor sites utilized upstream of the first coding exon, in the presence or the absence of noncoding exons between the major splice donor at the LTR and the splice acceptor at the first coding exons, and in the splicing pattern between the coding exons. Alternate splice acceptors were utilized between the coding exons of tat and rev, but the altered tat proteins did not differ in their ability to transactivate the SIV-LTR. The splicing for env mRNA is more complex than previously reported. Both singly and multiply spliced transcripts exist for env mRNA, and the same splice acceptor site is utilized by both rev and env mRNA.

Amino Acid Sequence↗
PubMed1992

The role of the tnv protein and tnv RNA splicing signals in replication of HIV-1 IIIB isolates.

The requirement for tnv, a tat-env-rev fusion protein expressed by the IIIB strain of HIV-1, was tested. The expression of tnv was prevented by altering the 5' splice site that flanks the central coding exon of tnv. Mutants that carry such an altered 5' splice site replicate normally in an established T-cell line and in peripheral blood lymphocytes, demonstrating that tnv has no effect on virus replication. However, two mutants that carry an alteration in the 3' splice site of the same exon are replication defective. The 3' splice site mutations result in significant reduction in the expression of the 16-kDa tat protein and induce the expression of large amounts of a 19-kDa rev-related protein that initiates within the central coding exon of tnv. S1 nuclease analysis reveals that splicing to the central tnv exon occurs with substantially increased efficiency via the use of an alternate 3' splice site six nucleotides 3' from the mutated site. The effect of the 3' splice site mutations on viral protein expression and replication are fully reversed by a second site mutation that eliminates the alternate splice site.

Base Sequence↗