Role of DEAD-box RNA helicase 1 in the mechanism of class switch recombination at the immunoglobulin heavy-chain locus

b细胞具有独特的能力，可以针对几乎无限的抗原产生量身定制的高度特异性抗体反应，这是由于抗体基因的多样化而成为可能。当B细胞分化为抗体分泌细胞时，基因缺失和重组事件在IgH位点发生，这种机制被称为类开关重组（CSR）。CSR将编码IgH链恒定区（CH外显子）的外显子交换为另一种抗体同型（例如IgG），从而定义抗体的效应函数。在分子水平上，CSR依赖于每个CH外显子上游启动的长链非编码RNA（开关RNA）进行重组的转录。由于开关RNA序列的g丰富度和高度重复性，这些转录本可以折叠成g -四重结构（G4 RNA），并可以稳定地与模板DNA链杂交形成r环结构。G4 RNA和R-loop结构都与靶向诱变酶激活诱导的胞苷脱氨酶（AID）到IgH位点，启动CSR有关。Ribeiro小组研究了RNA结合蛋白（rbp）如何调节Ig位点的DNA重组。特别是，我们对DEAD-box RNA解旋酶的作用感兴趣，这是一大家族的rbp，利用ATP水解来重塑RNA结构或RNA-蛋白质相互作用。Ribeiro等先前的研究表明，DEAD-box RNA解旋酶1 （DDX1）与G4 RNA结合并将其转化为Rloops，从而靶向AID并促进CSR （Ribeiro de Almeida et al., 2018）。本博士项目旨在了解DDX1和G4 RNA在AID靶向下游的作用。我们未发表的数据表明，DDX1参与了艾滋病诱导的DNA双链断裂（dsb）的DNA修复。重要的是，AID不恰当地靶向非ig热点（脱靶基因）和DSB异常修复可导致B细胞淋巴瘤的发生。因此，一个相关的问题是AID靶向的DDX1/G4 RNA机制是否与B细胞淋巴瘤发生有关。为了解决这些问题，我们将1)描述DDX1参与DSB修复的分子机制；2)研究DDX1是否与DSB修复途径选择和相关的基因组不稳定性有关；3)确定DDX1在CSR中的作用与免疫应答过程中b细胞活化的关系。这些研究将为rna介导的CSR控制机制提供新的见解，该机制的解除可能导致免疫疾病或b细胞淋巴瘤的发生。

PhD project strategic theme: Understanding the rules of lifeB cells have the unique ability to produce a tailored and highly specific antibody response against a virtually limitless range of antigens, which is possible due to diversification of antibody genes. As B cells differentiate into antibody secreting cells, gene deletional and recombinatorial events occur at the IgH locus by a mechanism known as class switch recombination (CSR). CSR exchanges the exon encoding the constant region of the IgH chain (CH exon) for an alternative antibody isotype (e.g. IgG), thereby defining the effector function of antibodies. At the molecular level, CSR depends on transcription of long non-coding RNA (switch RNA) initiated upstream of each CH exon undergoing recombination. Due to the G-richness and highly repetitive nature of switch RNA sequences, these transcripts can fold into G-quadruplex structures (G4 RNA) and can stably hybridize to the template DNA strand forming R-loop structures. Both G4 RNA and R-loop structures have been implicated in targeting the mutagenic enzyme activation-induced cytidine deaminase (AID) to the IgH locus, to initiate CSR.The Ribeiro group studies how RNA binding proteins (RBPs) regulate DNA recombination at Ig loci. In particular, we are interested in the role of DEAD-box RNA helicases, a large family of RBPs that utilize ATP hydrolysis to remodel RNA structures or RNA-protein interactions. The Ribeiro group have previously shown that DEAD-box RNA helicase 1 (DDX1) binds to G4 RNA and converts it into Rloops, thus targeting AID and promoting CSR (Ribeiro de Almeida et al., 2018). This PhD project aims to understand the role DDX1 and G4 RNA play downstream of AID targeting. Our unpublished data suggests DDX1 participates in the DNA repair of AID-induced DNA double-strand breaks (DSBs). Importantly, inappropriate targeting of AID to non-Ig hotspots (off-target genes) and aberrant DSB repair leads to the development of B cell lymphoma. Therefore, a related question is whether a DDX1/G4 RNA mechanism of AID targeting is associated with B cell lymphomagenesis. To address these questions, we will 1) characterize the molecular mechanisms by which DDX1 participates in DSB repair; 2) investigate if DDX1 is implicated in DSB repair pathway choice and associated genomeinstability; 3) determine how DDX1's role in CSR relates to B-cell activation during an immune response. These studies will provide new insight into RNA-mediated mechanisms controlling CSR, which deregulation can result in the development of immune disease or B-cell lymphomagenesis.