The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transition. The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transition

The B cell to plasma cell transition relies on the coordinated remodelling of the gene expression program. We performed a CRISPR/Cas9 knockout screen of RNA binding proteins to identify post-transcriptional regulation of gene expression during the B cell terminal differentiation. The m6A binding protein YTHDF2 was identified as promoting B cell terminal differentiation. In the absence of YTHDF2 the germinal centre reaction is broadly intact, however, plasma cells fail to accumulate. YTHDF2 has previously been shown to mediate RNA decay through the recruitment of the CCR4-NOT deadenylase machinery. Furthermore, our genetic screen in B cells identified that other CCR4-NOT recruitment factors inhibit rather than promote differentiation. Thus, the CCR4-NOT complex coordinates competing regulatory pathways of B cell differentiation at the post-transcriptional level. Overall design: m6A-eCLIP libraries were generated from two biological replicates in B cells after 8 days of iGB culture. Libraries from the input RNA were also generated for each of the replicates. For CRISPR screens, an sgRNA library targeting RNA binding proteins was transduced at day 3 in iGB culture. Screens were performed to assess proliferation/survival (guide representation at day 8 compared with day 4 in bulk B cells, 1 biological replicate each), and differentiation (guide representation in CD138+ compared with CD138- cells at day 8, 2 biological replicates each).

B细胞向浆细胞的转化依赖于基因表达程序的协同重塑。我们针对RNA结合蛋白开展了CRISPR/Cas9敲除筛选，以鉴定B细胞终末分化过程中基因表达的转录后调控机制。研究鉴定出m6A结合蛋白（m6A binding protein）YTHDF2可促进B细胞终末分化。在YTHDF2缺失的情况下，生发中心反应大体完整，但浆细胞无法正常积累。既往研究已证实，YTHDF2可通过招募CCR4-NOT脱腺苷酶复合物介导RNA降解。此外，我们在B细胞中开展的遗传筛选还发现，其他CCR4-NOT招募因子反而会抑制细胞分化，而非促进分化。综上，CCR4-NOT复合物在转录后水平协调了B细胞分化的多条竞争性调控通路。实验设计：于诱导性生发中心（inducible germinal centre, iGB）培养体系中培养8天后，从两份生物学重复的B细胞样本中构建m6A增强交联免疫沉淀（m6A-enhanced crosslinking immunoprecipitation, m6A-eCLIP）文库；同时为每份重复样本构建输入RNA对照文库。在CRISPR筛选实验中，我们于iGB培养第3天转导靶向RNA结合蛋白的单向导RNA（single guide RNA, sgRNA）文库。筛选实验分为两组：其一用于评估增殖与存活能力（比较批量B细胞在培养第8天与第4天的sgRNA代表丰度，各设置1份生物学重复）；其二用于评估分化能力（比较培养第8天时CD138阳性与CD138阴性细胞中的sgRNA代表丰度，各设置2份生物学重复）