RESUMO
Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm (5)) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, we show that eight mRNAs were increased in level by>2-fold in the pcm5 mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm (5) mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Exorribonucleases/metabolismo , Proteínas de Choque Térmico/genética , Discos Imaginais/metabolismo , MicroRNAs/metabolismo , Asas de Animais/embriologia , Animais , Drosophila/embriologia , Exorribonucleases/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/metabolismo , Discos Imaginais/embriologia , MicroRNAs/genética , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Asas de Animais/metabolismoRESUMO
Cordycepin (3' deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3' processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3' sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase α also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3' processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.
