RESUMO
To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3'-5' and/or XRN4 5'-3' exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome-mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.
Assuntos
Exonucleases/metabolismo , Exossomos , Proteínas de Membrana/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Interferência de RNA , Proteínas de Arabidopsis/genética , Códon de Iniciação/genética , Exonucleases/genética , Regulação da Expressão Gênica de Plantas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fases de Leitura Aberta , Proteínas de Plantas/genética , Estabilidade de RNA/genética , RNA de Plantas/genética , RNA Interferente Pequeno , Nicotiana/genéticaRESUMO
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry. Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporter mRNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient overexpression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.
Assuntos
Proteínas de Arabidopsis/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , RNA Helicases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Códon sem Sentido , RNA Helicases DEAD-box , Regulação da Expressão Gênica de Plantas , Humanos , Folhas de Planta/metabolismo , Proteínas Proto-Oncogênicas , RNA Helicases/genética , RNA Mensageiro , Homologia de Sequência , Nicotiana/genéticaRESUMO
When a ribosome reaches a stop codon, the eukaryotic Release Factor 1 (eRF1) binds to the A site of the ribosome and terminates translation. In yeasts and plants, both over- and underexpression of eRF1 lead to altered phenotype indicating that eRF1 expression should be strictly controlled. However, regulation of eRF1 level is still poorly understood. Here we show that expression of plant eRF1 is controlled by a complex negative autoregulatory circuit, which is based on the unique features of the 3Îuntranslated region (3ÎUTR) of the eRF1-1 transcript. The stop codon of the eRF1-1 mRNA is in a translational readthrough promoting context, while its 3ÎUTR induces nonsense-mediated decay (NMD), a translation termination coupled mRNA degradation mechanism. We demonstrate that readthrough partially protects the eRF1-1 mRNA from its 3ÎUTR induced NMD, and that elevated eRF1 levels inhibit readthrough and stimulate NMD. Thus, high eRF1 level leads to reduced eRF1-1 expression, as weakened readthrough fails to protect the eRF1-1 mRNA from the more intense NMD. This eRF1 autoregulatory circuit might serve to finely balance general translation termination efficiency.