Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.
ABSTRACT: Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.
Project description:In mammals, nonsense-mediated mRNA decay (NMD) functions in post-transcriptional gene regulation as well as mRNA surveillance. A key NMD factor, Upf1, becomes hyperphosphorylated by SMG1 kinase during the recognition of NMD substrates. Hyperphosphorylated Upf1 interacts with several factors including SMG5, SMG6, SMG7 and PNRC2 to trigger rapid mRNA degradation. However, the possible cross-talk among these factors and their selective use during NMD remain unknown. Here, we show that PNRC2 is preferentially complexed with SMG5, but not with SMG6 or SMG7, and that downregulation of PNRC2 abolishes the interaction between SMG5 and Dcp1a, a component of the decapping complex. In addition, tethering experiments reveal the function of Upf1, SMG5 and PNRC2 at the same step of NMD and the requirement of SMG6 for Upf1 for efficient mRNA degradation. Intriguingly, microarray results reveal the significant overlap of SMG5-dependent NMD substrates more with PNRC2-dependent NMD substrates than with SMG7-dependent NMD substrates, suggesting the functional dominance of SMG5-PNRC2, rather than SMG5-SMG7, under normal conditions. The results provide evidence that, to some extent, endogenous NMD substrates have their own binding preference for Upf1-interacting adaptors or effectors.
Project description:Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.
Project description:Analysis of cellular SMD or NMD substrates that regulated by Upf1 and/or PNRC2 in HeLa cell. The hypothesis tested in the present study was that endogenous SMD or NMD substrates may co-regulated by Upf1 and PNRC2. Results provide important information that vast range of cellular SMD or NMD substrates are reqired PNRC2 for decay. Overall design: Total RNA obtained from HeLa cells with downregulation of Upf1 or PNRC2 by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication
Project description: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.
Project description:Analysis of cellular SMD or NMD substrates that regulated by Upf1 and/or PNRC2 in HeLa cell. The hypothesis tested in the present study was that endogenous SMD or NMD substrates may co-regulated by Upf1 and PNRC2. Results provide important information that vast range of cellular SMD or NMD substrates are reqired PNRC2 for decay. Total RNA obtained from HeLa cells with downregulation of Upf1 or PNRC2 by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication
Project description:Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay.
Project description:Analysis of cellular NMD (Nonsense-mediated mRNA decay) substrates that regulated by Upf1, SMG5, SMG7 and/or PNRC2 in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates may co-regulated by Upf1, SMG5, SMG7 and PNRC2. Total RNA obtained from HeLa cells with downregulation of Upf1, SMG5, PNRC2 or SMG7 by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication
Project description:The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3'-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells.
Project description:The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein (CBP) 80 and 20. During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNAs increases in the cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is one of NMD-inducing features. Using chimeric reporter constructs and confocal microscopy, we find that the uORF of CDKN1A mRNA is actively translated and modulates a translational efficiency of the main downstream ORF. Our findings provide the biological insights into the possible role of NMD in diverse pathways mediated by CDKN1A. The microarray analysis performed to analize the cellular NMD substrates that regulated by Upf1, PNRC2 and/or CTIF in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates may co-regulated by Upf1, PNRC2 and CTIF. Results provide important information that vast range of cellular NMD substrates are reqired CTIF. Total RNA obtained from HeLa cells with downregulation of Upf1, PNRC2 or CTIF by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication.
Project description:Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms.