Unspliced Transcripts Research Articles

A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H.

An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3'-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 approximately 60 nucleotides downstream of 3'-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5' extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3'-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two U-to-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3'-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF(35) for binding to the exon sequence flanking 3'-splice site A3. This binding results in the inhibition of splicing at 3'-splice site A3.

An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of gag and pol, is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional. Viral proteins were identified in spleen tissue sections by immunohistochemistry and gp120 was present in splenic macrophages, T and B cells, and in serum. Clinical signs included wasting, mild to severe skin lesions, opaque cataracts, neurological signs, and respiratory difficulty. Histopathology included a selective loss of splenocytes within the periarterial lymphoid sheath, increased apoptosis of endothelial cells and splenocytes, follicular hyperplasia of the spleen, lymphocyte depletion of mesenteric lymph nodes, interstitial pneumonia, psoriatic skin lesions, and neurological, cardiac, and renal pathologies. Immunologically, delayed-type hypersensitivity response to keyhole limpet hemocyanin was diminished. By contrast, Ab titers and proliferative response to recall antigen (keyhole limpet hemocyanin) were normal. The HIV-1 Tg rat thus has many similarities to humans infected with HIV-1 in expression of viral genes, immune-response alterations, and pathologies resulting from infection. The HIV-1 Tg rat may provide a valuable model for some of the pathogenic manifestations of chronic HIV-1 diseases and could be useful in testing therapeutic regimens targeted to stages of viral replication subsequent to proviral integration.

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by HIV-1-based lentivirus vectors expressing transdominant Rev.

Retrovirus vectors expressing transdominant-negative mutants of Rev (TdRev) inhibit human immunodeficiency virus type 1 (HIV-1) replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env, and genomic RNA. The use of HIV-1-based vectors to express TdRev would have the advantage of allowing access to nondividing hematopoietic cells. It would also provide additional levels of protection by sequestering the viral regulatory proteins Tat and Rev, competing for encapsidation into wild-type virions, and inhibiting reverse transcription. Here we describe HIV-1-based vectors that express TdRev. These vectors contain mutations in the splicing signals or replacement of the Rev-responsive element by the simian retrovirus type 1 constitutive transport element, making them less sensitive to the inhibitory effects of TdRev. In addition, overexpression of Rev and the use of an HIV-1 helper plasmid that drives high levels of Gag-Pol synthesis were used to transiently overcome the inhibition by TdRev of the synthesis of Gag-Pol during vector production. SupT1 cells transduced with these vectors were more resistant to HIV-1 replication than cells transduced with Moloney murine leukemia virus-based vectors expressing TdRev. Furthermore, we show that these vectors can be mobilized by the wild-type virus, reducing the infectivity of virions escaping inhibition and conferring protection against HIV-1 replication to previously untransduced cells.

Expression of cardiotoxin‐2 gene

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)‐2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500‐bp (spliced) CTX‐2 mRNA as the final transcript. However, the liver produces two types of CTX‐2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP‐1, C/EBP, CACCC‐binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

Expression of cardiotoxin-2 gene. Cloning, characterization and deletion analysis of the promoter.

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

Translation is not required To generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells.

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as virion precursor RNA (vpRNA) for encapsidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate vpRNA. HIV-1-infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 Gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady-state Gag are competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell translation. Results of both [(3)H]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduces the absolute amounts of both cytoplasmic and virion-associated RNA. Evaluation of encapsidation efficiency by RPA revealed that the cytoplasmic availability of vpRNA is increased, indicating that HIV-1 unspliced mRNA can be rerouted to function as vpRNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as vpRNA.

The odyssey of a regulated transcript

The transcript of the Saccharomyces cerevisiae gene, RPL30, is subject to regulated splicing and regulated translation, due to a structure that interacts with its own product, ribosomal protein L30. We have followed the fate of the regulated RPL30 transcripts in vivo. Initially, these transcripts abortively enter the splicing pathway, forming an unusually stable association with U1 snRNP. A large proportion of the unspliced molecules, however, are found in the cytoplasm. Most of these are still bound by L30, as only a small fraction are engaged in translation. Eventually, the unspliced RPL30 transcripts escape the grasp of L30, associate with ribosomes, and fall prey to nonsense mediated decay.

Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus.

We have used a quantitative RNase protection assay to characterize the relative accumulation and abundance of individual adeno-associated virus type 2 (AAV) RNAs throughout the course of AAV-adenovirus coinfections and preinfections. We have demonstrated that there is a previously unrecognized temporal order to the appearance of AAV RNAs. First, unspliced P5-generated transcripts, which encode Rep78, were detectable prior to the significant accumulation of other AAV RNAs. Ultimately, as previously demonstrated, P19-generated products accumulated to levels greater than those generated from P5, and P40-generated transcripts predominated in the total RNA pool. Second, the percentage of each class of AAV RNA that was spliced increased during infection, and the degree of this increase was different for the P5/P19 products than for those generated by P40. At late times postcoinfection, approximately 90% of P40 products, but only approximately 50% of RNAs generated by P5 and P19, were seen to be spliced; thus, the AAV intron was removed to different final levels from these different RNA species. We have shown that each of the AAV RNAs is quite stable; the majority of each RNA species persisted 6 h after treatment with actinomycin D. Quantification of the accumulation of individual AAV RNAs, over intervals during which degradation was negligible, allowed us to infer that at late times during infection the relative strength of P5, P19, and P40 was approximately 1:3:18, respectively, consistent with the steady-state accumulated levels of the RNAs generated by each promoter. All AAV RNAs exited to the cytoplasm with similar efficiencies in the presence or absence of adenovirus; however, adenovirus coinfection appeared to stimulate total splicing of AAV RNAs and the relative use of the downstream intron acceptor. Our results confirm and extend previous observations concerning the appearance and processing of AAV-generated RNAs.

Antisense waxy genes with highly active promoters effectively suppress waxy gene expression in transgenic rice.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

A Novel Shuttle Protein Binds to RNA Helicase A and Activates the Retroviral Constitutive Transport Element

The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1.

Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation

AbstractX-linked chronic granulomatous disease (CGD) derives from defects in the CYBB gene, which encodes the gp91-phox component of NADPH oxidase. We studied the molecular basis of the disease in a kindred with variant CGD, due to a single base substitution at the sixth position of CYBB first intron. The patients’ phagocytes have been shown previously to greatly increase superoxide release in response to interferon-gamma (IFN-γ) in vitro and in vivo. We examined CYBB gene expression in an Epstein-Barr virus (EBV)-transformed B-cell line from 1 patient in this kindred. These cells showed markedly decreased levels of CYBB transcripts in total RNA (5% of normal) and nuclear RNA (1.4% of normal), despite equal CYBB transcription rates in the CGD and control cells. Incubation with IFN-γ produced a 3-fold increase in CYBBtotal messenger RNA (mRNA) levels in the patient’s cells, and decreased nuclear transcripts to undetectable levels. Reverse transcriptase–polymerase chain reaction analysis of RNA splicing revealed a preponderance of unspliced CYBB transcripts in the patient’s nuclear RNA. In vitro incubation with IFN-γ increased by 40% the ratio of spliced relative to unspliced CYBB mRNA in nuclei from the CGD B-cell line. Total RNA harvested from the same patient’s monocytes, on and off therapy with IFN-γ, showed a similar improvement in splicing. We conclude that IFN-γ partially corrects a nuclear processing defect due to the intronic mutation in theCYBB gene in this kindred, most likely by augmentation of nuclear export of normal transcripts, and improvement in the fidelity of splicing at the first intron.

Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation

X-linked chronic granulomatous disease (CGD) derives from defects in the CYBB gene, which encodes the gp91-phox component of NADPH oxidase. We studied the molecular basis of the disease in a kindred with variant CGD, due to a single base substitution at the sixth position of CYBB first intron. The patients' phagocytes have been shown previously to greatly increase superoxide release in response to interferon-gamma (IFN-γ) in vitro and in vivo. We examined CYBB gene expression in an Epstein-Barr virus (EBV)-transformed B-cell line from 1 patient in this kindred. These cells showed markedly decreased levels of CYBB transcripts in total RNA (5% of normal) and nuclear RNA (1.4% of normal), despite equal CYBB transcription rates in the CGD and control cells. Incubation with IFN-γ produced a 3-fold increase in CYBBtotal messenger RNA (mRNA) levels in the patient's cells, and decreased nuclear transcripts to undetectable levels. Reverse transcriptase–polymerase chain reaction analysis of RNA splicing revealed a preponderance of unspliced CYBB transcripts in the patient's nuclear RNA. In vitro incubation with IFN-γ increased by 40% the ratio of spliced relative to unspliced CYBB mRNA in nuclei from the CGD B-cell line. Total RNA harvested from the same patient's monocytes, on and off therapy with IFN-γ, showed a similar improvement in splicing. We conclude that IFN-γ partially corrects a nuclear processing defect due to the intronic mutation in theCYBB gene in this kindred, most likely by augmentation of nuclear export of normal transcripts, and improvement in the fidelity of splicing at the first intron.

Quantification of HIV-1 proviral DNA in patients with undetectable plasma viremia over long-term highly active antiretroviral therapy

Objectives: To assess the prognostic role of proviral DNA in peripheral blood mononuclear cells (PBMC) of patients with undetectable viremia over long-term highly active antiretroviral therapy (HAART). Methods: Eighty-two human immunodeficiency virus (HIV)-1-infected patients, free of acquired immunodeficiency syndrome (AIDS), received zidovudine plus lamivudine plus indinavir. Levels of plasma HIV-RNA, and PBMC proviral DNA and RNA unspliced (US) transcripts were evaluated by using competitive polymerase chain reaction (cPCR) assays, every 3 months over 1 year. Results: Among patients with undetectable viremia at baseline, 13 of 18 with CD4 cell count 350/mm 3 or less and 12 of 16 with CD4 between 351 and 700/mm 3, constantly maintained undetectable RNA levels; in these patients, a mean proviral DNA decrease of 0.67 ± 0.7 and 1.03 ± 0.53 log (P < 0.001), respectively, a significant decrease of RNA-US transcripts (P < 0.001), and significant correlations between decreases of proviral DNA and RNA-US transcripts (P = 0.008 and P < 0.001, respectively) were observed. Conclusions: Proviral DNA quantitation permits the continued monitoring of HAART in patients with undetectable viremia.

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo.

Structure and chromosomal location of the rat gene encoding the heart fatty acid-binding protein.

The gene coding for rat heart fatty acid-binding protein (FABP), along with 1.2 kb of its 5'-untranscribed region, was amplified by PCR, cloned and sequenced. As in other FABP genes, the coding sequence is interrupted by three introns of 3.4, 1.4 and 1.1 kb, respectively. Fluorescence in situ hybridization mapping revealed that the gene is located on chromosome 5q36. Using intron-specific primers flanking exon 2, unspliced primary transcript RNA of the FABP gene was detected in a preparation of total RNA isolated from rat heart, proving that the cloned gene is expressed in adult cardiac tissue.

Nucleic Acid Sequence-Based Amplification, a New Method for Analysis of Spliced and Unspliced Epstein-Barr Virus Latent Transcripts, and Its Comparison with Reverse Transcriptase PCR

Nucleic Acid Sequence-Based Amplification, a New Method for Analysis of Spliced and Unspliced Epstein-Barr Virus Latent Transcripts, and Its Comparison with Reverse Transcriptase PCR

Quantitative assessment of cell-associated and cell-free virus in cervicovaginal samples of HIV-1-infected women.

To examine the amount of cell-free and cell-associated virus in cervicovaginal secretions (CVS) of HIV-infected women. Paired cervicovaginal and blood samples from 61 seropositive women were quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcription-PCR (cRT-PCR) for: (1) genomic RNA from plasma and cell-free CVS, and (2) unspliced (u/s) RNA transcripts and proviral DNA in cells from secretions. HIV DNA was detected in 42.6%, u/s transcripts in 32.7% and cell-free HIV RNA in 31.1% of 61 cervicovaginal samples. The median copy numbers of HIV DNA, u/s transcripts, and cell-free RNA were 125 copies/10(5) cells, 40 copies/10(5) cells, and 300 copies/mL of secretion, respectively. Nineteen of 26 (73.1%) and 17 of 26 (65.3%) women positive for DNA were also positive for RNA transcripts and cell-free RNA, respectively (P<0.001). A significant correlation between the amounts of cell-free and u/s transcripts was also found (Spearman Rho 0.618, P=0.014). The prevalences of u/s transcripts and cell-free RNA were 42.6% and 53.8% respectively among patients with detectable blood RNA, and 22.9% (P=0.09) and 14.3% (P=0.0017) among patients with undetectable blood RNA. In stepwise logistic regression, cell-free RNA was independently associated with the presence of detectable blood viremia. The amount of HIV DNA was lower among subiects currently under treatment (50 copies/10(5) cells) than in untreated subjects (250 copies/10(5) cells) (P=0.037). Both cell-free and cell-associated HIV could be detected and quantitated in CVS, providing a means to examine the level of viral activity in the female genital tract.

Identification of false-positive CBFbeta/MYH11 RT-PCR results.

Persistent problems with false positive results were encountered when carrying out a published RT-PCR method to detect the CBFbeta/MYH11 transcripts associated with the inv(16)(p13q22) cytogenetic abnormality in acute myeloid leukaemia. These were shown to be due to amplification of part of the intronic MYH11 sequence, presumably from very small amounts of contaminating DNA or unspliced primary RNA transcripts, amplified because of partial homology of the CBFbeta3 primer to intronic MYH11 sequence.

SUBOPTIMAL SPLICE SIGNALS AND THE REX FUNCTION ARE IMPORTANT FOR THE CYTOPLASMIC ACCUMULATION OF INTRON-CONTAINING HTLV-1 mRNA

P111 The Rex protein of HTLV-1 is expressed early in the viral life cycle from doubly spliced mRNA. Rex mediates the transition to the late phase of viral gene expression which is characterized by structural protein translation from intron containing transcripts. Differential splicing of viral mRNA is a prerequisite for this regulation. Here we asked whether the HTLV splice sites determine the inefficient splicing of viral RNA and whether they are relevant for the regulation of viral gene expression by Rex. The test construct is derived from the 3′ half of the provirus. It produces spliced and unspliced transcripts which are regulated by Rex. The 5′ and three 3′ splice sites were mutated and the amounts of spliced and unspliced RNA (total, cytoplasmic) determined in presence and absence of Rex. The inactivation of the 5′ splice site resulted in reduced RNA stability. Conversion of the splice donor into the consensus sequence increased the splicing rate. This showed that the wild type splice site is suboptimal and counteracts efficient splicing. The inactivation of the dominant splice acceptor resulted in increased splicing at a splice acceptor located 12 nt downstream. This result suggests that the close proximity of two acceptor sites could contribute to inefficient splicing. Coexpression of Rex with the mutated test constructs changed the ratio of spliced to unspliced RNA. The comparison of mutants with increased splicing to wild type revealed that the relative cytoplasmatic amount of unspliced transcripts was reduced in the presence of Rex. Therefore suboptimal splice sites could be important for efficient Rex regulation.

Antisense morpholino oligonucleotide analog induces missplicing of C-myc mRNA.

A 28-mer morpholino oligonucleotide analog was designed to hybridize to 8 bases of intron 1 and extend 2 bases beyond the translation initiation codon in exon 2 of the unspliced c-myc RNA transcript. Delivery of this compound into human chronic myeloid leukemia KYO1 cells, by streptolysin O permeabilization, resulted in almost total ablation of the 65 kDa c-MYC protein expression for at least 24 hours after treatment. An unexpected band with SDS-PAGE electrophoretic mobility indicating a protein of about 47 kDa was apparent on the 24-hour western blots that were developed using antibodies that recognize MYC protein C terminal epitopes. No inhibition of the approximately 2400 nt c-myc mRNA expression was observed by northern hybridization, a result of the inability of morpholino analogs to direct the activity of ribonuclease H. In fact, high molecular weight c-myc RNA species were found to have accumulated in antisense-treated KYO1 cells. Control sense and scrambled antisense morpholino analogs did not inhibit MYC protein expression or induce the appearance of the anomalous RNA and protein bands. Molecular analyses by RT-PCR and sequencing revealed that the morpholino antisense effector had (1) inhibited splicing of the c-myc pre-mRNA, (2) induced missplicing of the pre-mRNA, and (3) inhibited translation of normal spliced c-myc mRNA. Identical results were obtained with acute promyelocytic leukemia, acute lymphoblastic leukemia, and histiocytic lymphoma cell lines.