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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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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. |
format | Online Article Text |
id | pubmed-6017670 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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