RNA-Seq mapping enabled quantitative analysis of gene expression differences between WT and rbm48 mutants in Zea mays (Maize).

Purpose: The goals of this study are to compare NGS-derived transcriptomes (RNA-Seq) derived from rbm48 mutant and WT maize endosperm tissue. Homozygous rbm48 mutants exhibit a seed defect and are seedling lethal. Maize Rbm48 is homologouse to a human protein (rbm48) that is thought o be invoilved in the splicing of minor (U12) introns. This study characterizes DEGs between rbm48 alleles umu1 and umu2 and their WT counterparts and serves to identify U12 introns exhibiting retention as evidenced by RNA-seq read density within U12 introns in rbm49 mutants relative to WT controls. Methods: Endosperm mRNA profiles from 16-18 days after pollination (DAP) normal and rbm48 kernels in the Zea mays W22 background were generated by deep RNA-Seq. Total RNA was prepared for 4 biological replicates of paired rbm48 and normal sibling pools. 4 total RNA samples of rbm48 umu1 and their WT controls, and 4 samples of rbm48 umu2 and their WT controls were obtained (16 total). Non strand specific Tru-Seq illumine libraries were constructed and sequenced on the Illumina HiSeq. The sequence reads that passed quality filters were aligned with GSNAP, read counts/gene were determined with the HTSeq-Count utility in the HTSeq package and DEG analysis performed with DESeq2. Counts to U2 and U12 introns were also assessed and intron retention was assessed by measuring the Percent Splice Out (PSO) proportions for individual introns between mutant and WT samples. (PSO) calculations are based on the established methods for examining exon skip proportions (PSI). Conclusions: Differential gene expression analysis of rbm48 identifies large-scale changes. Considering counts in introns, the major U2-type introns are largely unaffected with only 3-5% of introns in expressed genes have a ?PSO >20% By contrast, 65% and 53% of U12-type introns in rbm48-umu1 and rbm48-umu2, respectively, have a ?PSO >20% suggesting more than half of U12-type introns are retained in rbm48 mutants. These RNA-Seq data help demonstrate that rbm48 plays a role in splicing U12 introns. Overall design: Endosperm mRNA profiles from 16-18 days after pollination (DAP) normal and rbm48 kernels in the Zea mays W22 background were generated by deep RNA-Seq. Total RNA was prepared for 4 biological replicates of paired rbm48 and normal sibling pools (16 samples total). 2x100bp PE sequencing performed on an Illumina HiSeq2000.本研究旨在对比源自rbm48突变体与野生型（Wild Type, WT）玉米胚乳组织的下一代测序（Next-Generation Sequencing, NGS）转录组（RNA测序，RNA-Seq）数据。纯合rbm48突变体表现出种子缺陷且幼苗致死。玉米Rbm48与人类蛋白RBM48同源，后者被认为参与次要内含子（U12内含子，U12 intron）的剪接过程。本研究对rbm48等位基因umu1、umu2与其对应的野生型材料之间的差异表达基因（Differentially Expressed Genes, DEGs）进行了表征，并旨在鉴定相较于野生型对照，rbm48突变体中U12内含子区域的RNA-seq读段密度所体现出的内含子滞留现象。 方法：本研究以玉米（Zea mays）W22遗传背景下授粉后16-18天（Days After Pollination, DAP）的正常与rbm48突变籽粒为材料，通过深度RNA测序（RNA-Seq）构建胚乳mRNA表达谱。实验设置4组生物学重复，分别对rbm48突变体与正常姊妹株的混合样本进行配对分析；最终获得rbm48 umu1及其野生型对照样本各4份，rbm48 umu2及其野生型对照样本各4份，总计16份RNA样本。构建非链特异性Tru-Seq Illumina文库，并在Illumina HiSeq平台上完成测序。通过质量过滤的序列读段使用GSNAP软件进行比对，利用HTSeq软件包中的HTSeq-Count工具计算基因读段计数，随后借助DESeq2软件完成差异表达基因分析。同时对U2型与U12型内含子的计数进行评估，并通过计算突变体与野生型样本中单个内含子的剪接外显率百分比（Percent Splice Out, PSO）来评估内含子滞留情况。剪接外显率百分比（PSO）的计算基于用于分析外显子跳跃比例的经典方法——剪接保留指数（Percent Spliced In, PSI）。 结论：对rbm48突变体的差异表达基因分析显示其存在大规模基因表达变化。针对内含子区域的计数分析表明，主流的U2型内含子基本不受影响，在表达基因中仅有3%-5%的内含子的ΔPSO>20%。与之形成鲜明对比的是，rbm48-umu1与rbm48-umu2突变体中分别有65%与53%的U12型内含子的ΔPSO>20%，表明超过半数的U12型内含子在rbm48突变体中发生滞留。本RNA测序数据证实，rbm48参与U12型内含子的剪接过程。 整体实验设计：本研究以玉米（Zea mays）W22遗传背景下授粉后16-18天（DAP）的正常与rbm48突变籽粒为材料，通过深度RNA测序（RNA-Seq）构建胚乳mRNA表达谱。实验设置4组生物学重复，分别对rbm48突变体与正常姊妹株的混合样本进行配对分析，总计获得16份样本。采用Illumina HiSeq2000平台完成2×100bp双端测序。