SMG-8 and SMG-9, two novel subunits of the SMG-1 complex,regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed “SURF” that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.     

Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, eIF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link between the EJC and recognition of PTCs and triggers NMD.     

An unusual arrangement of two 14-3-3-like domains in the SMG5–SMG7 heterodimer is required for efficient nonsense-mediated mRNA decay

The nonsense-mediated mRNA decay (NMD) pathway triggers the rapid degradation of aberrant mRNAs containing premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5, SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like domains with phosphorylated UPF1, the central NMD effector. Recruitment of SMG5, SMG6, and SMG7 causes NMD target degradation. In this study, we report the crystal structure of the Caenorhabditis elegans SMG5–SMG7 complex. The 14-3-3-like phosphopeptide recognition domains of SMG5 and SMG7 heterodimerize in an unusual perpendicular back-to-back orientation in which the peptide-binding sites face opposite directions. Structure-based mutants and functional assays indicate that the SMG5–SMG7 interaction is conserved and is crucial for efficient NMD in human cells. Notably, we demonstrate that heterodimerization increases the affinity of the SMG5–SMG7 complex for UPF1. Furthermore, we show that the degradative activity of the SMG5–SMG7 complex resides in SMG7 and that the SMG5–SMG7 complex and SMG6 play partially redundant roles in the degradation of aberrant mRNAs. We propose that the SMG5–SMG7 complex binds to phosphorylated UPF1 with high affinity and recruits decay factors to the mRNA target through SMG7, thus promoting target degradation.     

The SMG5–SMG7 heterodimer directly recruits the CCR4–NOT deadenylase complex to mRNAs containing nonsense codons via interaction with POP2

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that detects aberrant mRNAs containing nonsense codons and induces their rapid degradation. This degradation is mediated by SMG6, an NMD-specific endonuclease, as well as the SMG5 and SMG7 proteins, which recruit general mRNA decay enzymes. However, it remains unknown which specific decay factors are recruited and whether this recruitment is direct. Here, we show that SMG7 binds directly to POP2, a catalytic subunit of the CCR4–NOT deadenylase complex, and elicits deadenylation-dependent decapping and 5′-to-3′ decay of NMD targets. Accordingly, a catalytically inactive POP2 mutant partially suppresses NMD in human cells. The SMG7–POP2 interaction is critical for NMD in cells depleted of SMG6, indicating that SMG7 and SMG6 act redundantly to promote the degradation of NMD targets. We further show that UPF1 provides multiple binding sites for decapping factors. These data unveil a missing direct physical link between NMD and the general mRNA decay machinery and indicate that NMD employs diverse and partially redundant mechanisms to ensure robust degradation of aberrant mRNAs.     

Genetic background affects relative nonsense mRNA accumulation in wild-type and<Emphasis Type="Italic"> upf</Emphasis> mutant yeast strains

The Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) pathway targets mRNAs with premature stop codons and some wild-type mRNAs for accelerated decay. Upf1p, Upf2p and Upf3p are required for NMD. NMD-targeted mRNAs are degraded rapidly in wild-type cells and stabilized in upf1, upf2 or upf3 mutants. We report here that the relative CYH2 pre-mRNA/mRNA accumulation is enhanced in cells derived from a W303 background, compared with a variety of commonly used strains. The enhanced CYH2 pre-mRNA accumulation phenotype results from a larger difference in mRNA half-lives in the W303 strains than two previously used strains. This phenotype can be selected in crosses and is also seen in upf2 and upf3 mutants. These results suggest there are genes that influence the efficiency of NMD and that yeast strains derived from the W303 background may be useful for measurement of abundance and half-lives of low abundance, short-lived NMD substrates.     

Splicing enhances translation in mammalian cells: an additional function of the exon junction complex

In mammalian cells, spliced mRNAs yield greater quantities of protein per mRNA molecule than do otherwise identical mRNAs not made by splicing. This increased translational yield correlates with enhanced cytoplasmic polysome association of spliced mRNAs, and is attributable to deposition of exon junction complexes (EJCs). Translational stimulation can be replicated by tethering the EJC proteins Y14, Magoh, and RNPS1 or the nonsense-mediated decay (NMD) factors Upf1, Upf2, and Upf3b to an intronless reporter mRNA. Thus, in addition to its previously characterized role in NMD, the EJC also promotes mRNA polysome association. Furthermore, the ability to stimulate translation when bound inside an open reading frame appears to be a general feature of factors required for NMD.     

Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation‐dependent manner

Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3′ untranslated region (UTR). Activity‐regulated cytoskeleton‐associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3′ UTR, it was thought that arc mRNA is a natural target of nonsense‐mediated mRNA decay (NMD). Here, we show that DTE in human arc 3′ UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity‐dependent degradation of the mRNA.     

MicroRNP‐mediated translational activation of nonadenylated mRNAs in a mammalian cell‐free system

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4‐NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let‐7‐microRNP‐mediated translational activation of nonadenylated target mRNAs in a mammalian cell‐free system, which contains over‐expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4‐1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)‐binding protein (PABP) interacting motif (PAM2) from the TNRC6B‐SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP‐mediated mRNA regulation.     

Mutations and polymorphisms in the gene encoding regulatory subunit type 1‐alpha of protein kinase A (PRKAR1A): an update

PRKAR1A encodes the regulatory subunit type 1‐alpha (RIα) of the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov ). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIα haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIα haploinsufficiency, but also by the expression of altered RIα protein, as proven by analysis of expressed mutations in the gene, consisting of aminoacid substitutions and in‐frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed—these are frame‐shifts in the 3′ end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation‐negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. Hum Mutat 31:369–379, 2010. Published 2010 Wiley‐Liss, Inc.     

Exome sequencing identifies UPF3B as the causative gene for a Chinese non‐syndrome mental retardation pedigree

Mental retardation (MR) is a group of common and complex disabilities affecting the central nervous system and appears before the period of brain developmental maturity. Recently, only 40% of genetic MR has been identified, however 60% remains unexplained. In this study, we applied exome sequencing to identify the mutation p.R430X in UPF3B gene in an MR pedigree, which was validated by Sanger sequencing and completely cosegregated within this family. UPF3B gene encodes a protein involved in nonsense‐mediated mRNA decay (NMD). By real‐time quantitative PCR, we detected the significant difference in the mRNA expression levels of the UPF3B and the classical NMD pathway target growth arrest and DNA‐damage‐inducible‐beta (GADD45B) between the patients and the controls. Our results directly implicated that the mutation p.R430X in UPF3B gene was the genetic etiology of the MR pedigree.     

Involvement of hypoxia‐inducible factor‐1 HiF(1α) in IgE‐mediated primary human basophil responses

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE‐mediated activation of primary human basophils results in protein accumulation of the α‐subunit of hypoxia‐inducible factor 1α (HIF‐1α), which is differentially regulated compared with signals controlling histamine release. HIF‐1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3‐kinase, were critical for IgE‐mediated accumulation of HIF‐1α, although the latter crucially affected degranulation. Abrogating HIF‐1α expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF‐1α protein alters IgE‐induced ATP depletion in basophils, thus also supporting the production of the pro‐allergic cytokine IL‐4.     

Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. We show that a novel phosphatidylinositol 3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of Caenorhabditis elegans smg-1, one of seven smg genes involved in NMD. hSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1, hSMG-1-DA, results in a marked suppression of the PTC-dependent beta-globin mRNA degradation; whereas that of wild-type hSMG-1 enhances it. We also show that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Taken together, we conclude that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.