蝦造血激素3'端非轉錄區居間調升激素的轉錄作用

Abstract

甲殼類動物的免疫系統主要依賴血球抵禦外來病原，從血球分泌的造血激素(Astakine)可以刺激造血組織生產血球細胞。 造血激素有兩種不同的mRNA轉錄型，差別在於3’端非轉錄區(3'UTR)的長度。短轉錄型的3’端非轉錄區則只有294個核苷酸，而長轉錄型的3’端非轉錄區長達965個核苷酸，長度是載碼區的2.64倍。使用定量PCR分別偵測兩種轉錄型在血球中的表現，發現在失血後長轉錄型的表現量顯著上升;另外稚蝦期的長轉錄型表現量較之於其他的發育時期也有顯著的上升。用西方點墨法偵測失血後草蝦血球的造血激素表現量，發現失血後造血激素蛋白質表現量的增加也和長轉錄型的表現相吻合，由此可推測造血激素的表現主要由長轉錄型負責。 除此之外，張等人(2013)揭露長轉錄型的3’端非編碼區第687到939號核苷酸內有一段序列可能形成結構。將這段可能形成結構的序列接在冷光基因的3’端後，冷光基因的表現量會有顯著的增加。進行核苷酸序列突變(nt723-728)，破壞轉譯促進區上的莖環結構，或點突變改變一個核苷酸(nt728)，冷光基因的表現量與控制組相比則沒有顯著差異，但改變莖環結構兩側核苷酸破壞結構上的序列(nt723-728, nt746-751)而不影響結構則冷光基因的表現量會有顯著的增加。證實長轉錄型的3’端非編碼區第687到939號核苷酸內的莖環結構能提高上游載碼區的蛋白質轉譯。 使用核糖核酸電泳遷移試驗(EMSA)發現加入蝦血球蛋白質萃取物後轉譯促進區的分子量的增加，推測這段結構能結合血球細胞內蛋白質，形成蛋白質核酸複合物。 但血球內的核酸結合蛋白仍需進一部定性。本研究顯示草蝦當失血或稚蝦發育期需要大量造血時，有可能透過長轉錄型造血激素3’端非轉錄區第687到939號核苷酸內的莖環結構，促進上游造血激素轉譯表現。

In crustaceans, astakine plays an essential role in innate immunity on account of its ability to stimulate hematopoiesis. There are two kinds of astakine mRNA transcript, which differ in the length of the 3’ untranslated region (3'UTR). The 3’UTR of astakine cloned by Soderhall et al. (2005) is only 294bp, but another 3'UTR cloned by Hsiao and Song (2010) is 965bp; this latter 3’UTR is 2.64 times longer than its open reading frame (ORF). We used Q-PCR to differentially target these two transcripts, and observed a rapid increase in the expression of the long transcript immediately after the withdrawal of hemolymph; in addition, the transcript level is higher at the juvenile developmental stage. Western blot was used to show that the expression of astakine protein exhibits a similar increase to that of the long transcript. These results suggest that the long transcript plays a major role in astakine translation. A possible structural sequence, Ast 3'UTR687-939nt, was reported in an earlier study (Chang et al., 2013); here, we showed that luciferase activity was significantly increased by the presence of downstream Ast 3'UTR687-939nt. Furthermore, the luciferase activity was restored to normal levels when the sequence (nt723-728) was mutated, or a single nucleotide site mutation (nt 728; predicted to cause collapse of the stem loop structure) was introduced. Notably, other mutations at both sides of the stem loop (nt723-728, nt746-751), which are not predicted to affect the stem loop structure, significantly increased upstream luciferase activity. This indicates that the stem loop structure in Ast 3'UTR687-939nt is a critical region for regulating upstream ORF expression. Using EMSA, we found that hemolytic RNA binding protein(s) exist in crustacean hemocytes, and these form an Ast 3’UTR687-939nt-protein complex. We isolated one of these UTR binding proteins, and determined its sequence by LC-Mass-Mass. The identified RNA binding protein(s) did not match any of the sequences in the (sparsely populated) shrimp protein database, and thus appears to be a previously undescribed protein. Further study will be undertaken to characterize the novel RNPs. In summary, we report here that the stem loop of Ast 3’UTR687-939nt can enhance upstream astakine expression during shrimp bleeding or during the juvenile developmental stage.