Rare occurrence of cryptic 5’ splice sites by downstream 3’ splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana
{"title":"Rare occurrence of cryptic 5’ splice sites by downstream 3’ splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana","authors":"Alvin Sanjaya, Ryo Nishijima, Yuki Fujii, Makoto Asano, Kotaro Ishii, Yusuke Kazama, Tomoko Abe, Makoto T. Fujiwara","doi":"10.3389/fpls.2024.1388040","DOIUrl":null,"url":null,"abstract":"Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5’-splice site) and AG (3’-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing <jats:italic>in planta</jats:italic>. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the <jats:italic>EGY1</jats:italic> gene in an Ar-ion-induced <jats:italic>egy1-4</jats:italic> allele of <jats:italic>Arabidopsis thaliana</jats:italic>. <jats:italic>egy1-4</jats:italic> has an AG-to-AC mutation in the 3′-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of <jats:italic>EGY1</jats:italic> revealed that while most wild-type <jats:italic>EGY1</jats:italic> mRNAs had a single splicing pattern, <jats:italic>egy1-4</jats:italic> mRNAs had multiple splicing defects. Almost half of <jats:italic>EGY1</jats:italic> transcripts showed ‘intron retention’ at intron 3, while the other half exhibited activation of 3’ cryptic splice sites either upstream or downstream of the original 3’-splice site. Unexpectedly, around 8% of <jats:italic>EGY1</jats:italic> transcripts in <jats:italic>egy1-4</jats:italic> exhibited activation of cryptic 5′-splice sites positioned upstream of the authentic 5’-splice site of intron 3. Whole genome resequencing of <jats:italic>egy1-4</jats:italic> indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5’-splice sites by downstream 3’-splice site/exon mutations <jats:italic>in planta</jats:italic>.","PeriodicalId":12632,"journal":{"name":"Frontiers in Plant Science","volume":null,"pages":null},"PeriodicalIF":4.1000,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Plant Science","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3389/fpls.2024.1388040","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5’-splice site) and AG (3’-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3′-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed ‘intron retention’ at intron 3, while the other half exhibited activation of 3’ cryptic splice sites either upstream or downstream of the original 3’-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5′-splice sites positioned upstream of the authentic 5’-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5’-splice sites by downstream 3’-splice site/exon mutations in planta.
期刊介绍:
In an ever changing world, plant science is of the utmost importance for securing the future well-being of humankind. Plants provide oxygen, food, feed, fibers, and building materials. In addition, they are a diverse source of industrial and pharmaceutical chemicals. Plants are centrally important to the health of ecosystems, and their understanding is critical for learning how to manage and maintain a sustainable biosphere. Plant science is extremely interdisciplinary, reaching from agricultural science to paleobotany, and molecular physiology to ecology. It uses the latest developments in computer science, optics, molecular biology and genomics to address challenges in model systems, agricultural crops, and ecosystems. Plant science research inquires into the form, function, development, diversity, reproduction, evolution and uses of both higher and lower plants and their interactions with other organisms throughout the biosphere. Frontiers in Plant Science welcomes outstanding contributions in any field of plant science from basic to applied research, from organismal to molecular studies, from single plant analysis to studies of populations and whole ecosystems, and from molecular to biophysical to computational approaches.
Frontiers in Plant Science publishes articles on the most outstanding discoveries across a wide research spectrum of Plant Science. The mission of Frontiers in Plant Science is to bring all relevant Plant Science areas together on a single platform.