scholarly journals New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

Biology ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 706
Author(s):  
Vita Šetrajčič Dragoš ◽  
Vida Stegel ◽  
Ana Blatnik ◽  
Gašper Klančar ◽  
Mateja Krajc ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Single Gene ◽  
Stk11 Gene ◽  
Uncertain Significance ◽  
Nbn Gene ◽  
Splicing Isoforms ◽  
Alternative Splicing Events ◽  
Long Range Pcr ◽  
Aberrant Rna

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

scholarly journals Quick and easy: 1-day-protocol for detection of normal alternative splicing events and aberrant spliceogenic transcripts with deep RNA sequencing

2021 ◽  
Author(s):  
Vita Šetrajčič Dragoš ◽  
Vida Stegel ◽  
Ana Blatnik ◽  
Gašper Klančar ◽  
Mateja Krajc ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Single Gene ◽  
Stk11 Gene ◽  
Hands On ◽  
Nbn Gene ◽  
Splicing Isoforms ◽  
Alternative Splicing Events ◽  
Aberrant Rna

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting all normal splicing isoforms of STK11 gene that were previously published. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetics variants. Due to the simplicity of our approach it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

scholarly journals Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum

BMC Genomics ◽  
2015 ◽  
Vol 16 (1) ◽  
pp. 54 ◽  
Author(s):  
Bin-Bin Xie ◽  
Dan Li ◽  
Wei-Ling Shi ◽  
Qi-Long Qin ◽  
Xiao-Wei Wang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
High Frequency ◽  
Trichoderma Longibrachiatum ◽  
Alternative Splicing Events

scholarly journals A comparative transcriptional landscape of two castor cultivars obtained by single-molecule sequencing comparative analysis

2020 ◽  
Author(s):  
Wei Zhou ◽  
Yaxing Zhou ◽  
Guoli Zhu ◽  
Yun Wang ◽  
Zhibiao He ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Comparative Analysis ◽  
Alternative Splicing ◽  
Rna Sequencing ◽  
Transcriptome Sequencing ◽  
Full Length ◽  
Transcriptional Analysis ◽  
Sequencing Analysis ◽  
Short Read ◽  
Alternative Splicing Events

AbstractBackground and ObjectivesCastor (Ricinus communis L.) is an important non-edible oilseed crop. Lm type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in inflorescence structures and leaf shapes. To better understand the mechanisums underling these differences at the molecular level, we performed comparative transcriptional analysis.Materials and MethodsFull-length transcriptome sequencing and short-read RNA sequencing were employed.ResultsA total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm type female strains and normal amphiprotic strains, respectively. In Lm female strain and normal amphiprotic strains 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed great different gene expression of common and unique transcription factors between the two cultivars. Meanwhile, functional analysis of isoform was conducted. Full-length sequences were used as a reference genome, and short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis.ConclusionsThe results revealed considerable difference and expression diversity between two cultivars, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two cultivars.HighlightUsing the full-length transcriptome sequencing technology, we performed comparative analysis of transcription factors of two castor cultivars, analyzed alternative splicing events, and identified their lncRNAs.

Strand-Specific Total RNA Sequencing Establishes the Complete Transcriptome and Alternative Splicing Repertoire in Diffuse Large B Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 864-864
Author(s):  
Adrienne Greenough ◽  
Andrea Moffitt ◽  
Dereje Jima ◽  
Jane Healy ◽  
Amee Patel ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
B Cell ◽  
Rna Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Total Rna ◽  
Large B Cell Lymphoma ◽  
Non Coding Rnas ◽  
Alternative Splicing Events ◽  
Large B Cell

Abstract Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL. Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors. Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL. Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. Disclosures Mann: Quiagen: Research Funding.

scholarly journals Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Suraya Muzafar ◽  
Ravi Datta Sharma ◽  
Abdul Haseeb Shah ◽  
Naseem A. Gaur ◽  
Ujjaini Dasgupta ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Alternative Splicing ◽  
Rna Sequencing ◽  
Single Gene ◽  
Drug Susceptibility ◽  
Protein Isoforms ◽  
Sequencing Data ◽  
Splice Isoforms ◽  
Content Type

ABSTRACT Alternative splicing (AS)—a process by which a single gene gives rise to different protein isoforms in eukaryotes—has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans, has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans. Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3, which contains a single intron. The sod3Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans. IMPORTANCE The emergence of resistance in Candida albicans, an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans, here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.

Analysis of alternative splicing events by RNA sequencing in the ovaries of Xiang pig at estrous and diestrous

2018 ◽  
Vol 119 ◽  
pp. 60-68 ◽  
Author(s):  
Liang-Ting Tang ◽  
Xue-Qin Ran ◽  
Ning Mao ◽  
Fu-Ping Zhang ◽  
Xi Niu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Alternative Splicing Events

scholarly journals Alternative splicing and the evolution of phenotypic novelty

2017 ◽  
Vol 372 (1713) ◽  
pp. 20150474 ◽  
Author(s):  
Stephen J. Bush ◽  
Lu Chen ◽  
Jaime M. Tovar-Corona ◽  
Araxi O. Urrutia
Keyword(s):  
Alternative Splicing ◽  
Gene Duplication ◽  
Single Gene ◽  
Morphological Diversity ◽  
Cell Types ◽  
Evolutionary Diversification ◽  
Population Sizes ◽  
Alternatively Spliced ◽  
Alternative Splicing Events ◽  
Multicellular Organisms

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Linking transcription, RNA polymerase II elongation and alternative splicing

2020 ◽  
Vol 477 (16) ◽  
pp. 3091-3104 ◽  
Author(s):  
Luciana E. Giono ◽  
Alberto R. Kornblihtt
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Polymerase Ii ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Single Gene ◽  
Regulation Of Gene Expression ◽  
Regulation Of Transcription ◽  
Stepping Stones ◽  
Eukaryotic Transcription

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic — such as promoter structure — and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

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