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Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing

Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenyla...

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Autores principales: Wang, Haoran, Wang, Mingxiu, Cheng, Qiang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017670/
https://www.ncbi.nlm.nih.gov/pubmed/29518015
http://dx.doi.org/10.3390/molecules23030608
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author Wang, Haoran
Wang, Mingxiu
Cheng, Qiang
author_facet Wang, Haoran
Wang, Mingxiu
Cheng, Qiang
author_sort Wang, Haoran
collection PubMed
description Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.
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spelling pubmed-60176702018-11-13 Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing Wang, Haoran Wang, Mingxiu Cheng, Qiang Molecules Article Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage. MDPI 2018-03-08 /pmc/articles/PMC6017670/ /pubmed/29518015 http://dx.doi.org/10.3390/molecules23030608 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wang, Haoran
Wang, Mingxiu
Cheng, Qiang
Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title_full Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title_fullStr Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title_full_unstemmed Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title_short Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing
title_sort capturing the alternative cleavage and polyadenylation sites of 14 nac genes in populus using a combination of 3′-race and high-throughput sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017670/
https://www.ncbi.nlm.nih.gov/pubmed/29518015
http://dx.doi.org/10.3390/molecules23030608
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