影響核醣核酸酶H表現以探討其於R-loops與抗體類型轉換重組之相關聯性

Abstract

在外來抗原刺激之下，B cell進行抗體類別轉換重組 (class switch recombination, CSR) 以分泌不同抗體型式，由原本分泌IgM轉換為IgA、IgG、IgE、IgD，有關於CSR如何完成詳細步驟還不是那麼清楚，因此實驗想探討誘發活化性胞嘧啶核苷脫胺酶 (activation-induced cytidine deaminase, AID) 如何參與標的步驟，其中之一假說為R-loops参與作為標的。為了探討R-loops在CSR機制所扮演之角色，本實驗藉由之方法為改變細胞內RNase H表現量。在in vitro情況，RNase H會降解RNA/DNA hybrid的RNA，因此會破壞R-loop結構。因此實驗過量表現E. coli RNase HI或是murine RNase H1於小鼠B細胞株 (CH12F3-2A)，另一方面建立穩定表現RNase H2 shRNA的細胞株，降低細胞中RNase H2表現，進而觀察細胞中CSR是否受到影響。 結果顯示過量表現RNase HI (1) 的CH12F3-2A中，R-loops模式 (pattern) 與出現的頻率並無差異，或許可解釋為何CSR頻率在過量表現RNase HI (1) 不受影響。RNase H2 knock down使RNase H2任一subunit表現降低時，CSR皆有不同程度增加，有可能因R-loops出現頻率增加而使CSR隨之增加，但仍需更多實驗分析詳細機制才可更加確認。

Upon antigen stimulation, B cells undergo class switch recombination (CSR) to switch from secreting IgM to other isotypes, such as IgA, IgG, IgE, IgD. However, the detailed mechanism of many steps involved in the recombination was unclear yet. This study focused on the activation-induced cytidine deaminase (AID) targeting step. One of the hypotheses for this is R-loops. In order to find out the role of R-loops in CSR, in this study, we altered the expression of RNase H in cells. RNase H degrades RNA in an RNA/DNA hybrid in vitro, thus RNase H could destroy R-loops in cells. Therefore, we would either over-express E.coli or murine RNase H1 in murine B cells (CH12F3-2A) or knock down RNase H2 expression in cells by shRNA. We would investigate the pattern and percentage of R-loops in these cells. In addition, the level of CSR is checked in these cells as well. Results indicated that in RNase HI(1)-overexpression cells, the pattern and the frequency of R-loops were not affected. It might explain the reason of the level of CSR was unchanged in cells. For knocking down expression of any subunit of RNase H2 in cells, the level of CSR increased. One of the possible reasons was that R-loops increased in cells and enhanced CSR. However, the detail mechanism for this needed further experiments to confirm.