{"title":"从 RNAseq 输入中预测可转座元件 (TE) 插入以及 TE 对果蝇大脑转录组中 RNA 剪接和基因表达的影响。","authors":"Md Fakhrul Azad, Tong Tong, Nelson C Lau","doi":"10.1186/s13100-024-00330-z","DOIUrl":null,"url":null,"abstract":"<p><p>Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.</p>","PeriodicalId":18854,"journal":{"name":"Mobile DNA","volume":null,"pages":null},"PeriodicalIF":4.7000,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11462757/pdf/","citationCount":"0","resultStr":"{\"title\":\"Transposable Element (TE) insertion predictions from RNAseq inputs and TE impact on RNA splicing and gene expression in Drosophila brain transcriptomes.\",\"authors\":\"Md Fakhrul Azad, Tong Tong, Nelson C Lau\",\"doi\":\"10.1186/s13100-024-00330-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.</p>\",\"PeriodicalId\":18854,\"journal\":{\"name\":\"Mobile DNA\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.7000,\"publicationDate\":\"2024-10-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11462757/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Mobile DNA\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1186/s13100-024-00330-z\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"GENETICS & HEREDITY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Mobile DNA","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s13100-024-00330-z","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
引用次数: 0
摘要
最近的研究表明,内含子中的可转座元件(Transposable Elements,TEs)经常剪接到主基因编码转录本中并改变其编码。为了重新研究TE插入蛋白编码基因转录本的频率,我们使用转座子插入和删除分析器(TIDAL)程序重新分析了果蝇神经元昼夜节律RNAseq数据集和长RNA蝇中脑RNAseq数据集。我们的 TIDAL 结果能够从 RNAseq 数据中预测出几个 TE 插入,这与之前发表的研究结果一致。但是,我们也发现了许多 TE 缺失调用中的差异,如主要支持 TE 内含子保留的读数,以及很少支持与 TE 剪接的嵌合 mRNA。然后,我们对TE-mRNA融合候选基因采用了严格的基因组DNA-PCR(gDNA-PCR)和RT-PCR程序,以了解有多少生物信息学预测可以得到验证。通过测试w1118菌株(其深层长RNAseq数据来源于该菌株)并与OreR菌株进行比较,23个TIDAL候选者中只有9个(
Transposable Element (TE) insertion predictions from RNAseq inputs and TE impact on RNA splicing and gene expression in Drosophila brain transcriptomes.
Recent studies have suggested that Transposable Elements (TEs) residing in introns frequently splice into and alter primary gene-coding transcripts. To re-examine the exonization frequency of TEs into protein-coding gene transcripts, we re-analyzed a Drosophila neuron circadian rhythm RNAseq dataset and a deep long RNA fly midbrain RNAseq dataset using our Transposon Insertion and Depletion Analyzer (TIDAL) program. Our TIDAL results were able to predict several TE insertions from RNAseq data that were consistent with previous published studies. However, we also uncovered many discrepancies in TE-exonization calls, such as reads that mainly support intron retention of the TE and little support for chimeric mRNA spliced to the TE. We then deployed rigorous genomic DNA-PCR (gDNA-PCR) and RT-PCR procedures on TE-mRNA fusion candidates to see how many of bioinformatics predictions could be validated. By testing a w1118 strain from which the deeper long RNAseq data was derived and comparing to an OreR strain, only 9 of 23 TIDAL candidates (< 40%) could be validated as a novel TE insertion by gDNA-PCR, indicating that deeper study is needed when using RNAseq data as inputs into current TE-insertion prediction programs. Of these validated calls, our RT-PCR results only supported TE-intron retention. Lastly, in the Dscam2 and Bx genes of the w1118 strain that contained intronic TEs, gene expression was 23 times higher than the OreR genes lacking the TEs. This study's validation approach indicates that chimeric TE-mRNAs are infrequent and cautions that more optimization is required in bioinformatics programs to call TE insertions using RNAseq datasets.
期刊介绍:
Mobile DNA is an online, peer-reviewed, open access journal that publishes articles providing novel insights into DNA rearrangements in all organisms, ranging from transposition and other types of recombination mechanisms to patterns and processes of mobile element and host genome evolution. In addition, the journal will consider articles on the utility of mobile genetic elements in biotechnological methods and protocols.