Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes

Conservation of Nonsense-Mediated mRNA Decay Complex Components Throughout Eukaryotic Evolution

Nonsense-mediated mRNA decay (NMD) is an essential eukaryotic process regulating transcript quality and abundance, and is involved in diverse processes including brain development and plant defenses. Although some of the NMD machinery is conserved between kingdoms, little is known about its evolution. Phosphorylation of the core NMD component UPF1 is critical for NMD and is regulated in mammals by the SURF complex (UPF1, SMG1 kinase, SMG8, SMG9 and eukaryotic release factors). However, since SMG1 is reportedly missing from the genomes of fungi and the plant Arabidopsis thaliana, it remains unclear how UPF1 is activated outside the metazoa. We used comparative genomics to determine the conservation of the NMD pathway across eukaryotic evolution. We show that SURF components are present in all major eukaryotic lineages, including fungi, suggesting that in addition to UPF1 and SMG1, SMG8 and SMG9 also existed in the last eukaryotic common ancestor, 1.8 billion years ago. However, despite the ancient origins of the SURF complex, we also found that SURF factors have been independently lost across the Eukarya, pointing to genetic buffering within the essential NMD pathway. We infer an ancient role for SURF in regulating UPF1, and the intriguing possibility of undiscovered NMD regulatory pathways

N- and C-terminal Upf1 phosphorylations create binding platforms for SMG-6 and SMG-5:SMG-7 during NMD

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors

Gene Variants Involved in Nonsense-Mediated mRNA Decay Suggest a Role in Autism Spectrum Disorder

This article belongs to the Special Issue mRNA Metabolism in Health and DiseaseAutism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition with unclear etiology. Many genes have been associated with ASD risk, but the underlying mechanisms are still poorly understood. An important post-transcriptional regulatory mechanism that plays an essential role during neurodevelopment, the Nonsense-Mediated mRNA Decay (NMD) pathway, may contribute to ASD risk. In this study, we gathered a list of 46 NMD factors and regulators and investigated the role of genetic variants in these genes in ASD. By conducting a comprehensive search for Single Nucleotide Variants (SNVs) in NMD genes using Whole Exome Sequencing data from 1828 ASD patients, we identified 270 SNVs predicted to be damaging in 28.7% of the population. We also analyzed Copy Number Variants (CNVs) from two cohorts of ASD patients (N = 3570) and discovered 38 CNVs in 1% of cases. Importantly, we discovered 136 genetic variants (125 SNVs and 11 CNVs) in 258 ASD patients that were located within protein domains required for NMD. These gene variants are classified as damaging using in silico prediction tools, and therefore may interfere with proper NMD function in ASD. The discovery of NMD genes as candidates for ASD in large patient genomic datasets provides evidence supporting the involvement of the NMD pathway in ASD pathophysiology.This research was supported by Fundação para a Ciência e a Tecnologia (UIDB/04046/2020 and UIDP/04046/2020 Centre grants to BioISI and PAC-POCI-01-0145-FEDER-016428 MEDPERSYST to A.M.V.) and by National Institute of Health Doutor Ricardo Jorge. A.R.M., J.V. and J.X.S. are recipients of a fellowship from BioSys PhD programme PD65-2012 (A.R.M. Ref: PD/BD/113773/2015; J.X.S. Ref: PD/BD/114386/2016; J.V. Ref: PD/BD/131390/2017) from Fundação para a Ciência e a Tecnologia (Portugal).info:eu-repo/semantics/publishedVersio

Structural and biochemical charaterization of the C. elegans SMG8-SMG9 core complex

Nonsense mediated mRNA decay (NMD) is an important mRNA quality control pathway conserved in eukaryotes. NMD targets aberrant mRNAs carrying premature stop codons (PTCs) for rapid degradation, preventing the accumulation of C-terminally truncated protein products that would otherwise be toxic to cells. NMD involves the concerted action of several trans-acting factors and it is a highly regulated process. A decisive event to trigger NMD in metazoans is the phosphorylation of the RNA helicase UPF1 by the SMG1 kinase. SMG8 and SMG9 form a heterodimer that interacts with SMG1 and inhibits its kinase activity. In recent years, electron microscopy studies of the human SMG1-SMG8-SMG9 complex provided low-resolution structural information that revealed the overall architecture of this complex. Still not much is known about the structure and function of SMG8 and SMG9 and how they interact with each other as well as with SMG1. In this thesis, I used biochemical approaches to identify the core of a SMG8-SMG9 complex amenable to crystallization and determined its three-dimensional structure at the resolution of 2.5 Å. I found that the C. elegans SMG8-SMG9 core complex resembles a G-domain heterodimer with a potentially active subunit (SMG9) and an inactive subunit (SMG8). Following this result, I characterized the nucleotide-binding properties of SMG-SMG9 using biophysical and structural methods. Fitting the atomic model in a previously published low-resolution EM map of a SMG1-SMG8-SMG9 complex raises interesting possibility that the nucleotide-binding state of SMG8-SMG9 might impact on the function of the kinase

The RNA helicase DHX34 functions as a scaffold for SMG1-mediated UPF1 phosphorylation

Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to promote mRNP remodelling, leading to activation of NMD. Here we demonstrate the mechanism by which DHX34 functions in concert with SMG1. DHX34 comprises two distinct structural units, a core that binds UPF1 and a protruding carboxy-terminal domain (CTD) that binds the SMG1 kinase, as shown using truncated forms of DHX34 and electron microscopy of the SMG1–DHX34 complex. Truncation of the DHX34 CTD does not affect binding to UPF1; however, it compromises DHX34 binding to SMG1 to affect UPF1 phosphorylation and hence abrogate NMD. Altogether, these data suggest the existence of a complex comprising SMG1, UPF1 and DHX34, with DHX34 functioning as a scaffold for UPF1 and SMG1. This complex promotes UPF1 phosphorylation leading to functional NMD

No-nonsense:insights into the functional interplay of nonsense-mediated mRNA decay factors

Nonsense-mediated messenger RNA decay (NMD) represents one of the main surveillance pathways used by eukaryotic cells to control the quality and abundance of mRNAs and to degrade viral RNA. NMD recognises mRNAs with a premature termination codon (PTC) and targets them to decay. Markers for a mRNA with a PTC, and thus NMD, are a long a 3′-untranslated region and the presence of an exon-junction complex (EJC) downstream of the stop codon. Here, we review our structural understanding of mammalian NMD factors and their functional interplay leading to a branched network of different interconnected but specialised mRNA decay pathways. We discuss recent insights into the potential impact of EJC composition on NMD pathway choice. We highlight the coexistence and function of different isoforms of up-frameshift protein 1 (UPF1) with an emphasis of their role at the endoplasmic reticulum and during stress, and the role of the paralogs UPF3B and UPF3A, underscoring that gene regulation by mammalian NMD is tightly controlled and context-dependent being conditional on developmental stage, tissue and cell types

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity

Structural and biochemical characterization of interactions centered on RNA decay factors: MTR4 and SMG1

The nuclear exosome is the central 3'-5' RNA degradation machinery that performs a myriad roles critical for the health of a cell. The exosome associates with the MTR4 helicase, which binds and unwinds RNA substrates that are threaded through the exosome barrel for degradation. In several cases, MTR4 is targeted to specific RNA substrates via its association with adaptor proteins. Since MTR4 is a component of several exosome adaptor complexes, it was hypothesized that it might be recognizing the adaptor proteins via a common motif. The first results section of this thesis presents a study in which I identified and characterized the interactions of the MTR4 helicase with a pre-ribosome processing adaptor, NVL and the scaffolding and MTR4 activating component of the nuclear exosome targeting complex, ZCCHC8. I identified that the N-terminal regions of NVL and ZCCHC8 contain conserved sequences resembling the arch interacting motif (AIM) of the yeast rRNA processing factors. The structural and biochemical analysis indicate that these AIM-like motifs bind the MTR4 arch domain in a manner similar to that of the AIMs described earlier in the literature. Overall, the results suggest that nuclear exosome adaptors have evolved canonical and non- canonical AIM sequences to bind to human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA- binding proteins. Recognizing RNA substrates for degradation is not only important in the nucleus but also in the cytoplasm. Nonsense mediated decay (NMD) is a cytoplasmic RNA decay mechanism which recognizes and degrades aberrant mRNA containing premature stop codons. It has also been shown to function in the regulation of physiological gene expression. SMG1, a 410 kDa PI3K related kinase, plays a crucial role in metazoan NMD by phosphorylating the UPF1 helicase. The phosphorylation of UPF1 was shown to be essential for the execution of NMD and represents the committed step of the NMD pathway. Although earlier low-resolution electron microscopic structures of human SMG1 along with some of its interacting partners were useful in gaining insight into the domain architecture of SMG1, the mechanism and regulation of SMG1 phosphorylation activity by SMG8-SMG9 remain poorly understood and are a subject of current research. The second results section presents a study, where I contributed to the characterization the C. elegans SMG8-SMG9 structurally and biochemically in an attempt to gain insights into the architecture of the complex and its possible biochemical role in NMD. The structure of the SMG8-SMG9 complex revealed that the complex exists as G-domain heterodimer with nucleotide binding capabilities. In a later study, presented as the third part of the results section, I contributed to understanding of the architecture of the SMG1-SMG8-SMG9 complex. The results not only recapitulate the findings of the SMG8-SMG9 complex but also provide structural basis for the SMG8-SMG9 interaction with SMG1. The structure also revealed that inositol-6-phosphate is a constitutive component of SMG1 and seems to play a role as a critical structural co-factor. The high- resolution structure of SMG1-SMG8-SMG9 provides a basis for several follow-up structural and biochemical studies centered on the early steps of NMD