Post-translational regulation of the exon skipping machinery controls aberrant splicing in T cell leukemia [Jurkat_H3B-8800 6hr]

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，儿科T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）存在异常剪接的替代调控机制，该机制通过去泛素化对剪接机器进行翻译后调控。整体实验设计：以1×10^6至5×10^6个T-ALL细胞系或原代细胞为材料，依照制造商说明书，使用Total RNA Mini Kit（Bio-Rad）提取总RNA。采用含寡聚脱氧胸苷的磁珠（Illumina）进行polyA+富集，或采用Ribominus RNA试剂盒进行核糖体RNA去除，以制备测序文库。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。将文库置于NextSeq 500/HiSeq 2500测序平台，采用76bp或50bp的双端读长策略进行测序。分别在两个细胞系（CUTLL1、Jurkat）的每个生物学重复与技术重复中，针对两组对比开展差异基因表达分析：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组对比实验：(a) 原代T细胞与T-ALL细胞，(b) 非高危型T-ALL与高危型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) 对照组与经SRSF6基因敲低的Jurkat细胞。所有分析均采用edgeR软件包完成。