scholarly journals Nuclear Assembly of UGA Decoding Complexes on Selenoprotein mRNAs: a Mechanism for Eluding Nonsense-Mediated Decay?

2006 ◽  
Vol 26 (5) ◽  
pp. 1795-1805 ◽  
Author(s):  
Lucia A. de Jesus ◽  
Peter R. Hoffmann ◽  
Tanya Michaud ◽  
Erin P. Forry ◽  
Andrea Small-Howard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Elongation Factor ◽  
Protein Translation ◽  
Nonsense Mediated Decay ◽  
Nuclear Assembly ◽  
Mrna Binding Protein ◽  
Two Factors ◽  
Mrna Binding ◽  
Selenoprotein Mrnas

ABSTRACT Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.

scholarly journals Supramolecular Complexes Mediate Selenocysteine Incorporation In Vivo

2006 ◽  
Vol 26 (6) ◽  
pp. 2337-2346 ◽  
Author(s):  
Andrea Small-Howard ◽  
Nadya Morozova ◽  
Zoia Stoytcheva ◽  
Erin P. Forry ◽  
John B. Mansell ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Elongation Factor ◽  
Supramolecular Complexes ◽  
Liver Protein ◽  
Mrna Levels ◽  
Nonsense Mediated Decay ◽  
Two Factors ◽  
Protein Nucleic Acid

ABSTRACT Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA[Ser]Sec and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA[Ser]Sec, termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA[Ser]Sec-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

scholarly journals Efficient Incorporation of Multiple Selenocysteines Involves an Inefficient Decoding Step Serving as a Potential Translational Checkpoint and RibosomeBottleneck

2006 ◽  
Vol 26 (24) ◽  
pp. 9177-9184 ◽  
Author(s):  
Zoia Stoytcheva ◽  
Rosa M. Tujebajeva ◽  
John W. Harney ◽  
Marla J. Berry
Keyword(s):  
Untranslated Region ◽  
Stop Codon ◽  
Secondary Structures ◽  
Selenoprotein P ◽  
Secis Element ◽  
Nonsense Mediated Decay ◽  
Slowing Down ◽  
Uga Codon ◽  
Sense Codon ◽  
Selenoprotein Mrnas

ABSTRACT Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a“ bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.

scholarly journals A genetically-encoded crosslinker screen identifies SERBP1 as a PKCε substrate influencing translation and cell division

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Silvia Martini ◽  
Khalil Davis ◽  
Rupert Faraway ◽  
Lisa Elze ◽  
Nicola Lockwood ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Translational Regulation ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Kinase Domain ◽  
Aurora B ◽  
Mrna Binding Protein ◽  
Genome Protection ◽  
Mrna Binding

AbstractThe PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection.

scholarly journals Selenium-Related Transcriptional Regulation of Gene Expression

2018 ◽  
Author(s):  
Mikko J. Lammi ◽  
Chengjuan Qu
Keyword(s):  
Stop Codon ◽  
Regulation Of Gene Expression ◽  
Selenium Deficiency ◽  
Mrna Levels ◽  
Translation Elongation ◽  
Nonsense Mediated Decay ◽  
Specific Complex ◽  
System Functioning ◽  
The One ◽  
Selenoprotein Mrnas

Selenium is a trace metal essential to human health, and its deficiency has been related to, for instance, cardiovascular and myodegenerative diseases, infertility and osteochondropathy Kashin-Beck disease. It is incorporated as selenocysteine to selenoproteins, which protect against reactive oxygen and nitrogen species. They also participate in the activation of thyroid hormone, and play a role in immune system functioning. The synthesis and incorporation of selenocysteine occurs via a special mechanism, which differs from the one used for standard amino acids. The codon for selenocysteine is the regular in-frame stop codon, which can be passed by specific complex machinery participating in translation elongation and termination. This includes the presence of selenocysteine insertion sequence (SECIS) in the 3’-untranslated part of the selenoprotein mRNAs. Selenium deficiency is known to control both selenoprotein and non-selenoprotein transcriptomes. Nonsense-mediated decay is involved in the regulation of selenoprotein mRNA levels, both other mechanisms are also possible.

scholarly journals THOC4 regulates energy homeostasis by stabilizing TFEB mRNA during prolonged starvation

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Toshiharu Fujita ◽  
Sayaka Kubo ◽  
Tatsuya Shioda ◽  
Ayaka Tokumura ◽  
Satoshi Minami ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Energy Homeostasis ◽  
Tail Length ◽  
Prolonged Starvation ◽  
Helix Loop Helix ◽  
Additional Layer ◽  
Lysosome Biogenesis ◽  
Mrna Binding Protein ◽  
Bhlh Transcription Factors ◽  
Mrna Binding

ABSTRACT TFEB, a basic helix-loop-helix transcription factor, is a master regulator of autophagy, lysosome biogenesis and lipid catabolism. Compared to posttranslational regulation of TFEB, the regulation of TFEB mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to TFEB mRNA and stabilized it after transcription by maintaining poly(A) tail length. We further found that this mode of regulation was conserved in Caenorhabditiselegans and was essential for TFEB-mediated lipid breakdown, which becomes over-represented during prolonged starvation. Taken together, our findings reveal the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function of TFEB in mediating autophagy and lipid metabolism.

scholarly journals Structures of translationally inactive mammalian ribosomes

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Alan Brown ◽  
Matthew R Baird ◽  
Matthew CJ Yip ◽  
Jason Murray ◽  
Sichen Shao
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Binding Protein ◽  
Elongation Factor ◽  
Regulate Gene Expression ◽  
Physiological Processes ◽  
Elongation Factor 2 ◽  
Mrna Binding Protein ◽  
Mrna Binding ◽  
Regulate Gene

The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the ‘Z site’, on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression.

scholarly journals Inhibition of eEF2K synergizes with glutaminase inhibitors or 4EBP1 depletion to suppress growth of triple-negative breast cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
YoungJun Ju ◽  
Yaacov Ben-David ◽  
Daniela Rotin ◽  
Eldad Zacksenhaus
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Translation Initiation ◽  
Triple Negative ◽  
Elongation Factor ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Related Factors ◽  
Metabolic Conditions

AbstractThe eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the ‘Collagen containing extracellular matrix’ pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

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