The RNA-binding protein Arpp21 interacts with and promotes Rag1 target mRNA expression to enable TCR rearrangement [RNA-seq]. The RNA-binding protein Arpp21 interacts with and promotes Rag1 target mRNA expression to enable TCR rearrangement [RNA-seq]

Post-transcriptional control by RNA-binding proteins (RBPs) is an essential layer of gene regulation in lymphocytes. Here, we show how members of the R3hdm RBP family regulate gene expression and promote the development of thymocytes. R3hdm paralogs with an intact R3H/SUZ domain bound mRNAs of other RBPs including Roquin-1, Roquin-2 and Nufip2, and augmented their expression. Arpp21 (R3hdm3) expression was restricted to thymocyte stages when Rag proteins recombine gene segments of the TCR variable region. Crosslinking and immunoprecipitation of Arpp21 in thymocytes revealed prominent binding to the Rag1 mRNA, and Arpp21 increased Rag1 3'-UTR reporter expression. Consequently, Arpp21–deficient mice showed decreased Rag1 expression in thymocytes, delayed TCR rearrangement, reduced frequencies of cells with TCR expression and a partial block of thymocyte development. The Arpp21 protein was regulated by Ca2+–signals that induced phosphorylation, polyubiquitination and proteasomal degradation. These findings involve general redundant functions of R3hdm RBP family proteins and show how stage-specific upregulation of Arpp21 in thymocytes promotes Rag1 expression preceding recombination while productive TCR rearrangement ceases this function. Overall design: Examination of transcriptome in WT or Arpp12 KO thymocytes. The Arpp21-CLIP experiment were performed on polyA+ RNA from thymocytes with 4 biological replicates.

RNA结合蛋白（RNA-binding proteins, RBPs）介导的转录后调控是淋巴细胞中基因调控的关键层级。本研究阐明了R3hdm家族RNA结合蛋白如何调控基因表达并促进胸腺细胞发育。带有完整R3H/SUZ结构域的R3hdm旁系同源蛋白，可结合包括Roquin-1、Roquin-2及Nufip2在内的其他RNA结合蛋白的mRNA，并上调其表达水平。Arpp21（即R3hdm3）的表达仅局限于Rag蛋白重组TCR可变区基因片段的胸腺细胞发育阶段。对胸腺细胞中的Arpp21进行交联免疫沉淀（crosslinking and immunoprecipitation, CLIP）实验发现，其可显著结合Rag1的mRNA，且Arpp21能够增强Rag1 3'-UTR报告基因的表达活性。相应地，Arpp21缺陷型小鼠的胸腺细胞中Rag1表达水平降低，TCR重排过程延迟，TCRβ阳性细胞的频率减少，且胸腺细胞发育存在部分阻滞。Arpp21蛋白的稳定性受钙离子信号调控：该信号可诱导Arpp21发生磷酸化、多泛素化并经蛋白酶体途径降解。本研究的发现既揭示了R3hdm家族RNA结合蛋白普遍存在的冗余功能，同时阐明了胸腺细胞阶段特异性上调的Arpp21，如何在TCR重排发生前促进Rag1表达，而当有效的TCR重排完成后，该调控功能则会终止。整体实验设计：对野生型（WT）或Arpp12敲除（KO）胸腺细胞的转录组进行检测；Arpp21-CLIP实验采用胸腺细胞来源的聚腺苷酸化RNA（polyA+ RNA），设置4个生物学重复。