Post-transcriptional Regulation of Genes Encoding Anti-microbial Peptides in Drosophila

Abstract

Secretion of antimicrobial peptides (AMPep) is a central defense mechanism used by invertebrates to combat infections. In Drosophila the synthesis of these peptides is a highly regulated process allowing their rapid release in the hemolymph upon contact with pathogens and the arrest of their production after pathogen clearance. We observed that AMPep genes have either a transient or sustained expression profile in S2 Drosophila cells treated with peptidoglycan. Moreover, AMPep genes containing AU-rich elements (ARE) in their 3-untranslated region (UTR) are subject to a post-transcriptional control affecting mRNA stability, thereby contributing to their transient expression profile. Cecropin A1 (CecA1) constitutes the prototype of this latter class of AMPeps. CecA1 mRNA bears in its 3-UTR an ARE similar to class II AREs found in several short-lived mammalian mRNAs. In response to immune deficiency cascade signaling activated by Gram-negative peptidoglycans, CecA1 mRNA is transiently stabilized and subsequently submitted to deadenylation and decay mediated by the ARE present in its 3-UTR. The functionality of CecA1 ARE relies on its ability to recruit TIS11 protein, which accelerates CecA1 mRNA deadenylation and decay. Moreover, we observed that CecA1 mRNA deadenylation is a biphasic process. Whereas early deadenylation is independent of TIS11, the later deadenylation phase depends on TIS11 and is mediated by CAF1 deadenylase. We also report that in contrast to tristetraprolin, its mammalian homolog, TIS11, is constitutively expressed in S2 cells and accumulates in cytoplasmic foci distinct from processing bodies, suggesting that the Drosophila ARE-mediated mRNA deadenylation and decay mechanism is markedly different in invertebrates and mammals.

dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

“…1B). As described previously (23), this degradation phase of the mRNA is concomitant with a further decrease in length of the remaining pool of messenger RNA (Fig. 1A, lanes 4 -8).…”

“…Recently, we and others have shown that AMD is conserved in Drosophila. In this organism, dTIS11 regulates the stability of ARE-containing mRNAs including messenger RNAs involved in the immune response, such as antimicrobial peptide-coding mRNAs (23)(24)(25). We have observed that AMD requires the CAF1 protein in Drosophila.…”

“…We have previously shown that this mRNA is deadenylated by a two-step mechanism (23). Our RNAi experiments indicated that both dTIS11 and CAF1 were necessary for the second phase of deadenylation, whereas depletion of these proteins alone had no detectable influence on the first phase of deadenylation (23).…”

“…However, as this kinetic of transcriptional shutdown cannot be precisely controlled, evaluations of CecA1 mRNA stability over short periods of time were performed in the presence of actinomycin D to achieve rapid transcriptional inhibition. Northern blot and RT-PCR were performed as described elsewhere (23). For high resolution Northern blot, RNA samples were separated by electrophoresis on 3% agarose gels.…”

“…28 and references therein). We isolated total mRNA from S2 cells induced by peptidoglycan for 6 h. As described previously (23), under these conditions, the initial deadenylation phase of CecA1 mRNA has been completed, and the remaining mRNA is engaged in the second phase of deadenylation and decay. To distinguish capped and decapped mRNAs, total RNA was either directly self-ligated with T4 RNA ligase or ligated after treatment with shrimp alkaline phosphatase followed by treatment with tobacco acid pyrophosphatase.…”