Post-transational regulation of the exon skipping machinery controls aberrant splicing in T cell leukemia [Jurkat_SI_24hr_300bp]

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. 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, 50bp or 151bp 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）存在异常剪接的另一套调控机制，即通过去泛素化作用对剪接机器进行翻译后调控。本研究从100万至500万个T-ALL细胞系或原代细胞中提取总RNA，操作严格遵循Total RNA Mini Kit（Bio-Rad）的制造商说明书。采用含寡聚dT的磁珠（Illumina）富集Poly-A+ RNA，或使用Ribominus RNA开展文库制备。使用TruSeq RNA样本制备试剂盒完成cDNA合成与非链特异性文库构建。文库在NextSeq 500/HiSeq 2500测序平台上进行测序，采用76bp、50bp或151bp的双端读长策略。分别针对两个细胞系（CUTLL1、Jurkat）的每一个生物学重复或技术重复，开展两组比较的差异基因表达分析：一是原代T细胞与T-ALL患者样本的对比，二是各配对处理组与对照组的对比。共设置7组比较方案：(a) 原代T细胞与T-ALL细胞；(b) 非高危T-ALL与高危T-ALL；(c) 对照组与经H3B-8800处理的Jurkat细胞；(d) 对照组与经H3B-8800处理的CUTLL1细胞；(e) 对照组与经P5091处理的Jurkat细胞；(f) 对照组与经P5091处理的CUTLL1细胞；(g) 对照组与SRSF6基因敲低的Jurkat细胞。差异分析采用edgeR软件包完成。