Post-translational regulation of the exon skipping machinery controls aberrant splicing in T cell leukemia [Jurkat_H3B-8800 24h]. Post-translational regulation of the exon skipping machinery controls aberrant splicing in T cell leukemia [Jurkat_H3B-8800 24h]

In this study, we show that pediatric T-cell acute lymphoblastic leukemia (T-ALL) has an alternative mechanism for aberrant splicing that involves post-translational regulation of the splicing machinery via deubiquitination. Overall design: Whole RNA was extracted from 1-5 million T-ALL (lines) cells or primary cells using the Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s protocol. Poly-A+ (magnetic oligodT-containing beads (Illumina)) or Ribominus RNA was used for library preparation. cDNA preparation and unstranded library construction was performed using the TruSeq RNA Sample Preparation Kit. Libraries were sequenced on the NextSeq 500/HiSeq 2500 using 76bp or 50bp paired-end read method. Differential gene expression analysis was performed for primary T cells vs T-ALL patients samples, or each matched treatment vs control pairs, separately in each biological or technical replicate in each of two cell lines (CUTLL1, Jurkat). Seven types of comparisons were tested: (a) T-Cells vs T-ALL, (b) Non high risk T-ALL vs high risk T-ALL, (c) Control vs H3B-8800 treated Jurkat cells, (d) Control vs H3B-8800 treated CUTLL1 cells, (e) Control vs P5091 treated Jurkat cells, (f) Control vs P5091 treated CUTLL1 cells, (g) Control vs SRSF6 knockdown Jurkat cells. Analysis was performed using edgeR package.

本研究证实，儿童T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）存在一种介导异常剪接的替代机制，该机制通过去泛素化对剪接装置进行翻译后调控。
实验设计：依照厂商说明书操作流程，使用Bio-Rad公司的Total RNA Mini Kit总RNA提取迷你试剂盒，从100万至500万个T-ALL细胞系或原代细胞中提取总RNA。采用依托Illumina公司含寡聚dT磁珠（magnetic oligodT-containing beads）富集得到的Poly-A+ RNA，或Ribominus RNA开展文库制备。使用TruSeq RNA Sample Preparation Kit完成cDNA制备及非链特异性文库构建。文库在NextSeq 500/HiSeq 2500测序平台上采用76bp或50bp双端读长策略进行测序。
针对原代T细胞与T-ALL患者样本，或各配对的处理组与对照组样本，分别在两个细胞系（CUTLL1、Jurkat）的每一个生物学重复及技术重复中开展差异基因表达分析。共设置7种比较组合：(a) T细胞与T-ALL样本；(b) 非高危型T-ALL与高危型T-ALL样本；(c) 对照组与H3B-8800处理的Jurkat细胞；(d) 对照组与H3B-8800处理的CUTLL1细胞；(e) 对照组与P5091处理的Jurkat细胞；(f) 对照组与P5091处理的CUTLL1细胞；(g) 对照组与SRSF6基因敲低的Jurkat细胞。所有分析均通过edgeR软件包完成。