HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [iCLIP-Seq]

Post-transcriptional regulation of mRNA by the RNA binding protein HuR is required in B cells for the germinal centre reaction and for the production of class-switched antibodies in response to T-independent antigens. Transcriptome-wide examination of RNA isoforms, abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interaction, revealed that HuR-dependent mRNA splicing affects hundreds of transcripts including the dihydrolipoyl succinyltransferase (Dlst), a subunit of the aketoglutaratedehydrogenase (aKGDH) enzyme. In the absence of HuR, defective mitochondrial metabolism results in high levels of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for B cell proliferation and differentiation. iCLIP analysis of HuR:RNA interaction in LPS-activated splenic B cells from C57BL/6 mice. Three biological replicates were sequenced using Illumina Genome Analyzer Iix platform.

RNA结合蛋白（RNA binding protein）HuR对mRNA的转录后调控，是B细胞发生生发中心反应、以及响应T非依赖抗原产生类别转换抗体所必需的。通过对HuR缺陷B细胞开展RNA异构体、表达丰度与翻译状态的全转录组检测，并结合HuR-RNA互作的直接检测分析，研究发现依赖HuR的mRNA剪接调控影响数百种转录本，其中包括二氢硫辛酰琥珀酰转移酶（dihydrolipoyl succinyltransferase, Dlst）——α-酮戊二酸脱氢酶（α-ketoglutarate dehydrogenase, αKGDH）的一个亚基。当HuR缺失时，线粒体代谢功能异常会导致活性氧（reactive oxygen species, ROS）水平升高，并引发B细胞死亡。本研究揭示了转录后调控过程如何维持B细胞增殖与分化所需的能量代谢平衡。针对C57BL/6小鼠脂多糖（lipopolysaccharide, LPS）激活的脾脏B细胞中HuR与RNA的互作进行iCLIP分析，采用Illumina Genome Analyzer Iix测序平台对3个生物学重复样本进行测序。