Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.
Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.
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DOI：
10.1371/journal.ppat.1006236
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发表时间：
2017-03
期刊：
影响因子：
6.7
通讯作者：
Kumar D
中科院分区：
文献类型：
--
作者：
Kalam H;Fontana MF;Kumar D
巨噬细胞在结核分枝杆菌（Mtb）感染时的转录重编程被广泛研究然而，交替剪接（AS）在形成对分枝杆菌感染的细胞反应中的重要性尚未得到重视。交替剪接可以影响转录物的稳定性或相应蛋白质的结构、功能和定位，从而改变蛋白质的化学计量和生理后果。通过对感染了Mtb毒株和毒株的人巨噬细胞的时间序列RNA-seq数据的综合分析，我们发现巨噬细胞转录组中有广泛的交替剪接重塑。这种调控的全球性是显而易见的，因为在感染的巨噬细胞中，属于运输、免疫反应、自噬、氧化还原和代谢等功能类的基因在剪接模式上表现出明显的差异。剪接机制在感染巨噬细胞中的系统性扰动是显而易见的，因为参与剪接体组装不同阶段的基因也在剪接水平上受到调节。奇怪的是，一些基因的截断/不可翻译变体的表达显著增加，特别是在毒性感染时。截断转录本的表达增加与相应蛋白水平的下降相关。我们验证了其中一个候选基因RAB8B的生理相关性；其截断的变体在H37Rv感染的细胞中富集。通过特殊的转导调节RAB8B转录本的较长或较短变体的相对丰度，可以分别促进或阻断针对溶酶体的分枝杆菌，从而导致细菌存活率的相应变化。我们的研究结果表明，RAB8B募集到分枝杆菌吞噬体是吞噬体成熟所必需的。因此，截断的RAB8B变体的丰度通过限制细胞中的RAB8B水平来帮助毒性Mtb存活，我们随后在人原代巨噬细胞中验证了这一机制。综上所述，我们证明了交替剪接作为结核分枝杆菌干预的新位点，并为开发抗结核分枝杆菌的新药物靶点提供了有吸引力的选择。真核生物基因表达是一个复杂的过程，在翻译发生之前需要转录物的几个中间处理步骤。基因表达的变化是细胞适应环境信号或刺激（如感染）的基本手段。专业吞噬细胞巨噬细胞在炎症刺激或结核分枝杆菌等病原体感染时基因表达发生剧烈变化。然而，基因表达的改变是否也可能受到RNA加工中一个或多个中间步骤的扰动的影响尚不清楚。在这里，利用下一代RNA测序方法，然后进行强大的计算分析，我们发现结核分枝杆菌感染巨噬细胞导致宿主剪接模式的大量改变。由于交替剪接可以影响转录物的稳定性、翻译产物的稳定性、功能的丧失/获得、相互作用的伴侣和亚细胞定位，因此它对宿主对感染的反应可能是压倒性的。我们随后进行了靶向实验，以证实宿主基因的交替剪接变体确实有助于感染结核分枝杆菌菌株在巨噬细胞内更好地生存。因此，来自宿主和/或病原体的剪接调节因子构成了一组有吸引力的目标，以开发新的治疗策略来控制结核病。
Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb) infection is widely studied; however, the significance of alternate splicing (AS) in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the cells, a mechanism which we subsequently verified in human primary macrophages. Taken together we demonstrate alternate splicing as a new locus of intervention by Mtb and provide attractive alternative to exploit for novel drug targets against Mtb. Eukaryotic gene expression is a complex process where several intermediary processing steps of transcripts are required before translation can take place. Change in gene expression is a fundamental means through which cells adapt to environmental cues or stimuli like infections. The professional phagocytes macrophages are known to undergo drastic alteration in gene expression upon inflammatory stimuli or infection with pathogens like Mycobacterium tuberculosis. However, whether the alterations in gene expression are also influenced by possible perturbations of one or more of the intermediate steps in RNA processing is not known. Here taking advantage of next-generation RNA sequencing approach, followed by robust computational analysis, we show infection of macrophages by Mycobacterium tuberculosis results in massive alterations in the splicing pattern in the host. Since alternate splicing can influence transcript stability, stability of the translated products, loss/gain of function, interacting partners and sub-cellular localization, its implications on host response to infection could be overwhelming. We subsequently performed targeted experiments to confirm that alternate spliced variants of host genes indeed helped infecting Mtb strains to survive better within the macrophages. Regulators of splicing, either from host and/or pathogen, therefore, constitute an attractive set of targets to develop novel therapeutic strategies to control tuberculosis.