A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy

1992 ◽  
Vol 90 (3) ◽  
Author(s):  
Yukihiko Mashima ◽  
RichardG. Weleber ◽  
NancyG. Kennaway ◽  
George Inana
Keyword(s):  
Rna Splicing ◽  
Base Change ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Ornithine Aminotransferase ◽  
Splice Acceptor ◽  
Gyrate Atrophy ◽  
Single Base ◽  
Single Base Change ◽  
Ornithine Aminotransferase Gene

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 Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
Xiang-xing Zhu ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

A single-base change at a splice site in Wx-A1 caused incorrect RNA splicing and gene inactivation in a wheat EMS mutant line

2019 ◽  
Vol 132 (7) ◽  
pp. 2097-2109 ◽  
Author(s):  
Mi Luo ◽  
Jinjin Ding ◽  
Yu Li ◽  
Huaping Tang ◽  
Pengfei Qi ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Base Change ◽  
Gene Inactivation ◽  
Mutant Line ◽  
Single Base ◽  
Single Base Change

A single-base change at a splice site in a β0-thalassemic gene causes abnormal RNA splicing

Cell ◽  
1982 ◽  
Vol 29 (3) ◽  
pp. 903-911 ◽  
Author(s):  
Richard Treisman ◽  
Nicholas J. Proudfoot ◽  
Monica Shander ◽  
Tom Maniatis
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Base Change ◽  
Single Base ◽  
Single Base Change

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
XIANGXING ZHU ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Denaturing Gradient Gel Analysis of Single-Base Substitutions at a MouseAdenine Phosphoribosyltransferase Splice Acceptor Site

1989 ◽  
Vol 2 (4) ◽  
pp. 217-225 ◽  
Author(s):  
Stephen R. Dlouhy ◽  
Dennis A. Schaff ◽  
James A. Trofatter ◽  
Hsiao-Sheng Liu ◽  
Peter J. Stambrook ◽  
...  
Keyword(s):  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Substitutions

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.

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