Identification of seven exonic variants in the SLC4A1, ATP6V1B1 and ATP6V0A4 gene that alter RNA splicing by minigene assay.

2021 ◽  
Author(s):  
Ruixiao Zhang ◽  
Zeqing Chen ◽  
Qijing Song ◽  
Sai Wang ◽  
Zhiying Liu ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Minigene Assay

scholarly journals Identification of eight exonic variants in the SLC4A1, ATP6V1B1 and ATP6V0A4 gene that alter RNA splicing by minigene assay

2021 ◽  
Author(s):  
Ruixiao Zhang ◽  
Zeqing Chen ◽  
Qijing Song ◽  
Sai Wang ◽  
Zhiying Liu ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Mrna Level ◽  
Exon Skipping ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Splicing Regulatory Elements ◽  
Renal Tubular ◽  
Splicing Site ◽  
Minigene Assay

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1 or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the pre-mRNA splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 8 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1( c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108*; c.1571C>T, p.Pro524Leu and c.1572G>A, p.Pro524Pro) were identified to result in whole or part of exon skipping by either disruption of ESEs and generation of ESSs, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro

scholarly journals Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress

2021 ◽  
Vol 22 (10) ◽  
pp. 5154
Author(s):  
Elena Putscher ◽  
Michael Hecker ◽  
Brit Fitzner ◽  
Peter Lorenz ◽  
Uwe Klaus Zettl
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Region Of Interest ◽  
Restriction Enzymes ◽  
Genomic Region ◽  
Gateway Cloning ◽  
Splicing Pattern ◽  
Processing Step ◽  
Alternative Splicing Events ◽  
Minigene Assay

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.

scholarly journals LAMB2 novel variant c.2885‐9 C>A affects RNA splicing in a minigene assay

2021 ◽  
Author(s):  
Xiaoyuan Wang ◽  
Huijie Xiao ◽  
Baige Su ◽  
Yali Ren ◽  
Jie Ding ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Novel Variant ◽  
Minigene Assay

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals Identification of the cross-strand chimeric RNAs generated by fusions of bi-directional transcripts

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuting Wang ◽  
Qin Zou ◽  
Fajin Li ◽  
Wenwei Zhao ◽  
Hui Xu ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Sequencing Data ◽  
Normal Tissues ◽  
Rna Transcripts ◽  
Chimeric Rnas ◽  
Dna Strands ◽  
Comprehensive Survey ◽  
The Cross ◽  
Chimeric Rna ◽  
Cancerous Cells

AbstractA major part of the transcriptome complexity is attributed to multiple types of DNA or RNA fusion events, which take place within a gene such as alternative splicing or between different genes such as DNA rearrangement and trans-splicing. In the present study, using the RNA deep sequencing data, we systematically survey a type of non-canonical fusions between the RNA transcripts from the two opposite DNA strands. We name the products of such fusion events cross-strand chimeric RNA (cscRNA). Hundreds to thousands of cscRNAs can be found in human normal tissues, primary cells, and cancerous cells, and in other species as well. Although cscRNAs exhibit strong tissue-specificity, our analysis identifies thousands of recurrent cscRNAs found in multiple different samples. cscRNAs are mostly originated from convergent transcriptions of the annotated genes and their anti-sense DNA. The machinery of cscRNA biogenesis is unclear, but the cross-strand junction events show some features related to RNA splicing. The present study is a comprehensive survey of the non-canonical cross-strand RNA junction events, a resource for further characterization of the originations and functions of the cscRNAs.

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