ABSTRACT
The term "nonsense-mediated mRNA decay" (NMD) originally described the degradation of mRNAs with premature translation-termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%-10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5-SMG7 or SMG5-PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5'-3' exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.
Subject(s)
Carrier Proteins/metabolism , Nonsense Mediated mRNA Decay/genetics , RNA Helicases/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Trans-Activators/metabolism , Carrier Proteins/genetics , Cell Line, Tumor , Codon, Nonsense/genetics , Gene Expression Regulation/genetics , HeLa Cells , Humans , Protein Binding/genetics , RNA Helicases/genetics , RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Trans-Activators/genetics , Two-Hybrid System TechniquesABSTRACT
Breast and ovarian cancers remain a major public health issue, constituting a major cause of female morbidity and mortality worldwide. Even though systemic chemotherapy remains the main course of action for both types of cancer, current research studies aim at the discovery of novel therapeutic targets. Ribonucleases, due to their apparent implication in gene expression regulation, may serve as potential anticancer drugs. Human RNase κ is an endoribonuclease belonging in a recently identified family of proteins. Recent data from expression microarray studies reveal that the RNase κ gene is found either up- or downregulated in a number of human cancers, indicating a possible diagnostic and/or prognostic utility.The aim of this study was to investigate modulations in the expression levels of RNase κ gene, in breast and ovarian cancer cells, as a response to treatment with different chemotherapeutic agents. BT-20 breast cancer cells and SKOV-3 ovarian cancer cells were treated with the cytotoxic drugs paclitaxel, docetaxel, cisplatin, carboplatin, epirubicin and vinorelbine. Gene expression analysis was performed by the comparative CT method also known as 2(-ΔΔCT) method. The results revealed a distinct increase in the expression levels of RNase κ mRNA (up to 9-fold) after treatment with the antineoplastic agent paclitaxel in both cell lines, while treatment with the remaining anticancer drugs did not alter drastically the mRNA levels of RNase κ. Based on the fact that paclitaxel exerts its cytotoxic action by inducing apoptosis, the results could be indicative of a potential implication of RNase κ in apoptosis-related pathways.
