scholarly journals Functional Mapping of the Human Papillomavirus Type 16 E1 Cistron

2008 ◽  
Vol 82 (21) ◽  
pp. 10724-10734 ◽  
Author(s):  
Michael J. Lace ◽  
James R. Anson ◽  
Lubomir P. Turek ◽  
Thomas H. Haugen
Keyword(s):  
Human Papillomavirus ◽  
Donor Site ◽  
Dna Amplification ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Mrna Levels ◽  
Viral Dna ◽  
Hpv 16 ◽  
Hpv Dna

ABSTRACT Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1− mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5′ mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1− genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.

Efficacy of DNA versus RNA NGS-based Methods in MET Exon 14 skipping mutation detection.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9036-9036
Author(s):  
Magdalena Jurkiewicz ◽  
Anjali Saqi ◽  
Mahesh M Mansukhani ◽  
Vaishali Hodel ◽  
Anamaria Krull ◽  
...  
Keyword(s):  
Splice Site ◽  
Donor Site ◽  
Driver Mutations ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Female Ratio ◽  
Lung Carcinomas ◽  
Rna Analysis ◽  
Lung Adenocarcinomas

9036 Background: Exon 14 skipping mutations in the mesenchymal-epithelial transition ( MET) gene are reported in 2-5% of lung adenocarcinomas and are mutually exclusive of other driver mutations. Small-molecule MET tyrosine kinase inhibitors, capmatinib and tepotinib, showed durable responses in previously treated and treatment-naïve patients harboring MET-exon-14 skipping mutations. Studies suggest that for detection of MET-ex14 mutations, DNA-based assays alone may be sub-optimal when compared to RNA-based NGS assays. We compared the performance of DNA and RNA-based assays for detection of MET-ex14 variants. Methods: We examined NGS-based profiling data of lung adenocarcinomas (or when this diagnosis could not be excluded) to identify MET-ex14 mutations missed by DNA but identified by RNA analysis. The carcinomas were profiled by a DNA-based NGS panel that targets MET exons 2, 14, 16, 18 and 19. Cases without driver mutations were reflexed to an NGS-based RNA fusion panel (Archer’s Anchored Multiplex PCR). Results: Over a 21-month period, MET-ex14 skipping events were detected in 16/644 (2.5%) lung carcinomas by DNA profiling. RNA analysis on driver-negative cases identified 9 additional MET-ex14 mutations. All 16 MET-ex14 DNA variants occurred at or around the intron 14 splice donor site, as the assay did not include the intron 13 splice acceptor site. Clinical characteristics of the MET positive cohort include a male to female ratio of 0.8:1.0, an average age of 76.5 years and 52% non-smoker status. All tumors were adenocarcinomas (including one with a < 10% spindle/pleomorphic component) with the exception of 3 adenosquamous carcinomas and 1 squamous cell carcinoma. Conclusions: DNA based NGS-panels can potentially miss MET-ex14 skipping events in lung carcinomas, when the primers do not target both 3′ splice site of intron 13, and the 5′ splice site of intron 14. A reflex work flow interrogating RNA fusions can potentially capture such events. The clinical and molecular characterization of the variants detected only by RNA NGS assays warrants further exploration.

scholarly journals Contribution of Noncanonical Splice Variants to TTN Truncating Variants Cardiomyopathy

2021 ◽  
Author(s):  
Parth N. Patel ◽  
Kaoru Ito ◽  
Jon A.L. Willcox ◽  
Alireza Haghighi ◽  
Min Young Jang ◽  
...  
Keyword(s):  
Splice Variants ◽  
Donor Site ◽  
Idiopathic Dilated Cardiomyopathy ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Variants Of Unknown Significance ◽  
Clinical Variant ◽  
Unknown Significance

Background: Heterozygous TTN truncating variants cause 10% to 20% of idiopathic dilated cardiomyopathy (DCM). Although variants which disrupt canonical splice signals (ie, invariant dinucleotide of splice donor site, invariant dinucleotide of the splice acceptor site) at exon-intron junctions are readily recognized as TTN truncating variants, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain. Methods: Rare variants of unknown significance located in the splice regions of highly expressed TTN exons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay. Results: Splice-altering variants of unknown significance were enriched in DCM cases over controls and present in 2% of DCM patients ( P =0.002). Application of this method to clinical variant databases demonstrated 20% of similar variants of unknown significance in TTN splice regions affect splicing. Noncanonical splice-altering variants were most frequently located at position +5 of the donor site ( P =4.4×10 7 ) and position -3 of the acceptor site ( P =0.002). SpliceAI, an emerging in silico prediction tool, had a high positive predictive value (86%–95%) but poor sensitivity (15%–50%) for the detection of splice-altering variants. Alternate exons spliced out of most TTN transcripts frequently lacked the consensus base at +5 donor and −3 acceptor positions. Conclusions: Noncanonical splice-altering variants in TTN explain 1-2% of DCM and offer a 10-20% increase in the diagnostic power of TTN sequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternate TTN exons.

Control of retroviral RNA splicing through maintenance of suboptimal processing signals

1990 ◽  
Vol 10 (2) ◽  
pp. 696-704 ◽  
Author(s):  
R A Katz ◽  
A M Skalka
Keyword(s):  
Rna Splicing ◽  
Donor Site ◽  
Protein S ◽  
Full Length ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Insertion Mutation ◽  
Acceptor Site ◽  
Splice Acceptor

The full-length retroviral transcript serves as genomic RNA for progeny virions, as an mRNA for structural proteins and enzymes, and as a pre-mRNA substrate for splicing that yields subgenomic mRNAs that encode other essential proteins. Thus, RNA splicing to form subgenomic mRNAs must be incomplete or regulated in order to preserve some of the full-length transcripts. We have used the avian sarcoma virus system to delineate the viral functions that are required in the regulation of the splicing event that forms the envelope glycoprotein (env) subgenomic mRNA. We observed previously that a specific insertion mutation just 5' of the env splice acceptor site resulted in nearly complete splicing to form env mRNA and a concomitant replication defect which is presumably due to a deficit of the full-length transcript. Replication-competent pseudorevertants contained second-site mutations that restored splicing control, and these mapped either just upstream or downstream of the env splice acceptor site. In this report, we show that splicing control at this site does not require expression of any known viral replication protein(s), nor does it appear to require the viral splice donor site. From these results and analysis of additional splicing mutations obtained by in vivo selection, we conclude that splicing is controlled through the maintenance of suboptimal cis-acting signals in the viral RNA that alter the efficiency of recognition by the cellular splicing machinery.

scholarly journals Human Cytomegalovirus 5-Kilobase Immediate-Early RNA Is a Stable Intron

2004 ◽  
Vol 78 (23) ◽  
pp. 13182-13189 ◽  
Author(s):  
Caroline A. Kulesza ◽  
Thomas Shenk
Keyword(s):  
Viral Infection ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Donor Site ◽  
Immediate Early ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Viral Gene ◽  
Acceptor Site ◽  
Splice Donor

ABSTRACT Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5′ end of the 5-kb RNA to a consensus splice donor site and localized the 3′ end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5′ splice donor site that defines the 5′ end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV.

scholarly journals Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

2001 ◽  
Vol 171 (3) ◽  
pp. 397-402 ◽  
Author(s):  
HH Lee ◽  
SF Chang
Keyword(s):  
Donor Site ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Site Mutation ◽  
Hydroxylase Deficiency ◽  
Exon 2 ◽  
Eukaryotic Genes ◽  
21 Hydroxylase Deficiency ◽  
Intron 2

Maturation of primary RNA transcripts of eukaryotic genes often involves the removal of introns and joining of exons. The fidelity of RNA splicing is dependent on the identity of the nucleotide (nt) sequences at exon/intron boundaries. Most importantly, the highly conserved intronic 5'GT and 3'AG sequences are essential for correct splicing. Substitution of GT by any other nt leads to incomplete mRNA and a disruption of protein structure. We describe here the results of our transfection experiments in COS-1 cells with a CYP21 genomic construct that contained an IVS 2+1G-->A mutation. Analysis of the transcripts by RT-PCR revealed that two different transcripts were generated by this mutant genome. In all the splicing products, we found that the entire exon 2 was deleted. Surprisingly, 30% of the transcripts from this mutant CYP21 genome were accompanied by an inclusion of 3' intron 2 sequences due to the use of a different splice acceptor site. This is the first report of the molecular characterization of a splice donor site mutation in CYP21 via transcription in COS-1 cells.

scholarly journals iSS-PseDNC: Identifying Splicing Sites Using Pseudo Dinucleotide Composition

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Wei Chen ◽  
Peng-Mian Feng ◽  
Hao Lin ◽  
Kuo-Chen Chou
Keyword(s):  
Structural Properties ◽  
Rna Splicing ◽  
Donor Site ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Sites ◽  
Success Rates ◽  
Eukaryotic Genes ◽  
Dinucleotide Composition

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called “iSS-PseDNC” was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called “pseudo dinucleotide composition” (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing.

scholarly journals Control of retroviral RNA splicing through maintenance of suboptimal processing signals.

1990 ◽  
Vol 10 (2) ◽  
pp. 696-704 ◽  
Author(s):  
R A Katz ◽  
A M Skalka
Keyword(s):  
Rna Splicing ◽  
Donor Site ◽  
Protein S ◽  
Full Length ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Insertion Mutation ◽  
Acceptor Site ◽  
Splice Acceptor

The full-length retroviral transcript serves as genomic RNA for progeny virions, as an mRNA for structural proteins and enzymes, and as a pre-mRNA substrate for splicing that yields subgenomic mRNAs that encode other essential proteins. Thus, RNA splicing to form subgenomic mRNAs must be incomplete or regulated in order to preserve some of the full-length transcripts. We have used the avian sarcoma virus system to delineate the viral functions that are required in the regulation of the splicing event that forms the envelope glycoprotein (env) subgenomic mRNA. We observed previously that a specific insertion mutation just 5' of the env splice acceptor site resulted in nearly complete splicing to form env mRNA and a concomitant replication defect which is presumably due to a deficit of the full-length transcript. Replication-competent pseudorevertants contained second-site mutations that restored splicing control, and these mapped either just upstream or downstream of the env splice acceptor site. In this report, we show that splicing control at this site does not require expression of any known viral replication protein(s), nor does it appear to require the viral splice donor site. From these results and analysis of additional splicing mutations obtained by in vivo selection, we conclude that splicing is controlled through the maintenance of suboptimal cis-acting signals in the viral RNA that alter the efficiency of recognition by the cellular splicing machinery.

scholarly journals Human Papillomavirus DNA Replication Compartments in a Transient DNA Replication System

1999 ◽  
Vol 73 (2) ◽  
pp. 1001-1009 ◽  
Author(s):  
C. Scott Swindle ◽  
Nianxiang Zou ◽  
Brian A. Van Tine ◽  
George M. Shaw ◽  
Jeffrey A. Engler ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Active Sites ◽  
Protein A ◽  
Dna Amplification ◽  
Brdu Incorporation ◽  
Viral Dna ◽  
Virion Assembly ◽  
Hpv Dna ◽  
Nuclear Foci

ABSTRACT Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.

scholarly journals Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

2010 ◽  
Vol 84 (16) ◽  
pp. 8219-8230 ◽  
Author(s):  
Monika Somberg ◽  
Stefan Schwartz
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Binding Sites ◽  
Mrna Levels ◽  
Coding Region ◽  
Cervical Lesions ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

scholarly journals Incorporation of Cervista Human Papillomavirus 16/18 Assay Into Algorithms for Classifying Human Papillomavirus Status in Formalin-Fixed, Paraffin-Embedded Head and Neck Squamous Carcinoma Specimens

2018 ◽  
Vol 143 (3) ◽  
pp. 356-361
Author(s):  
Ming Guo ◽  
Abha Khanna ◽  
Jianping Wang ◽  
Michelle D. Williams ◽  
Neda Kalhor ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Human Papillomavirus ◽  
Ffpe Tissue ◽  
Hpv 16 ◽  
Hpv Dna ◽  
Formalin Fixed Paraffin ◽  
Hpv Status ◽  
Formalin Fixed Paraffin Embedded ◽  
Hpv Assays ◽  
Formalin Fixed

Context.— Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. Objective.— To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. Design.— Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)–based HPV16/18 genotyping tests in cases with discrepancy. Results.— Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. Conclusions.— The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

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