Up-regulating translation of astakine, shrimp hematopoietic cytokine, mediated by its 3''UTR
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.
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