RESUMO
While disorders in lipid metabolism have been associated with aging and age-related diseases, how lipid metabolism is regulated during aging is poorly understood. Here, we characterize the Drosophila endoribonuclease CG2145, an ortholog of mammalian EndoU that we named Age-related lipid regulator (Arlr), as a regulator of lipid homeostasis during aging. In adult adipose tissues, Arlr is necessary for maintenance of lipid storage in lipid droplets (LDs) as flies age, a phenotype that can be rescued by either high-fat or high-glucose diet. Interestingly, RNA-seq of arlr mutant adipose tissues and RIP-seq suggest that Arlr affects lipid metabolism through the degradation of the mRNAs of lipolysis genes - a model further supported by the observation that knockdown of Lsd-1, regucalcin, yip2 or CG5162, which encode genes involved in lipolysis, rescue the LD defects of arlr mutants. In addition, we characterize DendoU as a functional paralog of Arlr and show that human ENDOU can rescue arlr mutants. Altogether, our study reveals a role of ENDOU-like endonucleases as negative regulator of lipolysis.
Assuntos
Endorribonucleases , Lipólise , Animais , Humanos , Lipólise/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Endorribonucleases Específicas de Uridilato/metabolismo , Metabolismo dos Lipídeos/genética , Drosophila/genética , Drosophila/metabolismo , Envelhecimento/genética , Lipídeos , Homeostase/genética , Gotículas Lipídicas/metabolismo , Mamíferos/metabolismoRESUMO
Upstream open reading frames (uORFs) are known to negatively affect translation of the downstream ORF. The regulatory proteins involved in relieving this inhibition are however poorly characterized. In response to cellular stress, eIF2α phosphorylation leads to an inhibition of global protein synthesis, while translation of specific factors such as CHOP is induced. We analyzed a 105-nt inhibitory uORF in the transcript of human CHOP (huORFchop ) and found that overexpression of the zebrafish or human ENDOU poly(U)-endoribonuclease (Endouc or ENDOU-1, respectively) increases CHOP mRNA translation also in the absence of stress. We also found that Endouc/ENDOU-1 binds and cleaves the huORFchop transcript at position 80G-81U, which induces CHOP translation independently of phosphorylated eIF2α. However, both ENDOU and phospho-eIF2α are nonetheless required for maximal translation of CHOP mRNA. Increased levels of ENDOU shift a huORFchop reporter as well as endogenous CHOP transcripts from the monosome to polysome fraction, indicating an increase in translation. Furthermore, we found that the uncapped truncated huORFchop -69-105-nt transcript contains an internal ribosome entry site (IRES), facilitating translation of the cleaved transcript. Therefore, we propose a model where ENDOU-mediated transcript cleavage positively regulates CHOP translation resulting in increased CHOP protein levels upon stress. Specifically, CHOP transcript cleavage changes the configuration of huORFchop thereby releasing its inhibition and allowing the stalled ribosomes to resume translation of the downstream ORF.
Assuntos
RNA Mensageiro/genética , Fator de Transcrição CHOP/genética , Endorribonucleases Específicas de Uridilato/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Motivos de Nucleotídeos , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Fator de Transcrição CHOP/metabolismo , Peixe-ZebraRESUMO
We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/transmissão , Placenta/enzimologia , Complicações na Gravidez/metabolismo , Endorribonucleases Específicas de Uridilato/metabolismo , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Gravidez , Mapas de Interação de Proteínas , SARS-CoV-2 , Análise de Sequência de RNARESUMO
Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.
