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.

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 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 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.

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.

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 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.

scholarly journals A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

2000 ◽  
Vol 74 (8) ◽  
pp. 3709-3714 ◽  
Author(s):  
Jérôme Déjardin ◽  
Guillaume Bompard-Maréchal ◽  
Muriel Audit ◽  
Thomas J. Hope ◽  
Marc Sitbon ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Leukemia Virus ◽  
Donor Site ◽  
Point Mutations ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Coding Region ◽  
Virus Rna ◽  
Leukemia Viruses ◽  
Murine Leukemia

ABSTRACT Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.

MO039IDENTIFICATION OF A RECURRENT SYNONYMOUS GENETIC VARIANT IN NPHP3 LEADING TO NEPHRONOPHTHISIS AND CONGENITAL HEPATIC FIBROSIS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Eric Olinger ◽  
Intisar Al Alawi ◽  
Elisa Molinari ◽  
Eissa Ali Faqeih ◽  
Mohamed Al Hamed ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Rna Splicing ◽  
Donor Site ◽  
Saudi Arabian ◽  
Bioinformatic Analysis ◽  
Initial Assessment ◽  
Splice Donor Site ◽  
Uk Biobank ◽  
Pathogenic Variants ◽  
Exon 20

Abstract Background and Aims Whole exome sequencing (WES) is becoming part of routine clinical and diagnostic practice and has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants. Filtering of variants and scoring variants in terms of pathogenicity still represents a major challenge and may explain why ∼50% of patients remain without diagnosis after initial assessment. Method In this study, we performed WES to determine the genetic cause of a hepato-renal ciliopathy syndrome in a genetically unsolved consanguineous family from Oman with 2 affected children. For variants filtering and annotation Qiagen Clinical Insight tool was used. Database searches for identical alleles in patients with similar phenotypes were performed using Genomics England, UK Biobank and a Saudi Arabian cohort. RNA studies were used to confirm a splicing defect. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project and from UK Biobank, a major biomedical database. Results Initial bioinformatic analysis of WES data from 2 affected sibs excluded obvious pathogenic variants in known genes associated with primary ciliopathy syndromes with liver and kidney phenotypes. However, by manual curation of variants in candidate genes, a rare homozygous synonymous allele in NPHP3 was identified (c.2805C&gt;T; p.(Gly935Gly)), mid-exon 20 and within a region of shared homozygosity on chromosome 3. Correct segregation was confirmed via Sanger sequencing in the parents and the 2 affected sibs. The variant was rare in gnomAD (2/251374 alleles) and was found heterozygously in just one individual within the UK Biobank cohort of 200,000 exomes. Using various in silico tools, the allele was shown to activate a cryptic splice donor site in the middle of exon 20. RT-PCR with sequencing of parental whole blood RNA confirmed alternative splicing leading to frameshift p.Gly935GlyfsTer47. The identical homozygous allele was identified in 2 additional unsolved families within the Genomics England 100,000 Genomes Project and in 1 Saudi Arabian family with similar hepato-renal phenotypes. Conclusion This study shows that automated filtering of WES data may exclude synonymous variants which are pathogenic, especially if they are mid-exon. Here we identified a recurrent synonymous NPHP3 variant leading to a splice defect as the cause of a hepato-renal ciliopathy phenotype in four families. In unsolved cases, rare synonymous alleles in candidate genes need to be reassessed for impact on RNA splicing and possible pathogenicity.

scholarly journals Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

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