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https://www.readbyqxmd.com/read/29277545/in-vitro-reconstitution-of-translational-arrest-pathways
#1
Qing Feng, Sichen Shao
Protein translation is tightly regulated to ensure high-fidelity expression of genetic information. Various conditions cause ribosomes to stall while synthesizing new proteins. Different types of translational arrest initiate specific mRNA surveillance, protein quality control, and stress response pathways that directly impact gene expression and protein homeostasis. Our understanding of these pathways is greatly enhanced by reconstituting these processes in cell-free systems. The high degree of biochemical manipulability of in vitro systems facilitates the identification of key machineries, mechanistic dissection of their functional roles, and structural analysis of intermediate complexes...
December 22, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29211986/trna-fluctuations-observed-on-stalled-ribosomes-are-suppressed-during-ongoing-protein-synthesis
#2
Ryan M Jamiolkowski, Chunlai Chen, Barry S Cooperman, Yale E Goldman
The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation...
December 5, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/29203495/pa5470-counteracts-antimicrobial-effect-of-azithromycin-by-releasing-stalled-ribosome-in-pseudomonas-aeruginosa
#3
Jing Shi, Yiwei Liu, Yueying Zhang, Yongxin Jin, Fang Bai, Zhihui Cheng, Shouguang Jin, Weihui Wu
Pseudomonas aeruginosa causes various acute and chronic infections in human. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosa infections. By binding to the exit tunnel of 50S ribosome, AZM causes ribosome stalling and depletion of intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosa cells and repress quorum sensing regulated virulence factors as well as swarming motility. In P. aeruginosa, PA5470 encodes a putative peptide chain release factor, whose expression is highly induced by macrolide antibiotics...
December 4, 2017: Antimicrobial Agents and Chemotherapy
https://www.readbyqxmd.com/read/29203248/vms1-a-cytosolic-cat-tailing-antagonist-to-protect-mitochondria
#4
Stefan G Kreft, Elke Deuerling
In eukaryotes, a cytosolic ribosome quality control complex recycles erroneously stalled ribosomes and modifies faulty nascent chains by ubiquitination and by C-terminal Ala- and Thr-extension (CAT-tailing). Reported recently in Cell, Izawa et al. identify cytosolic Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive 1) as an inhibitor of CAT-tailing, which prevents mitochondrial dysfunction caused by imported CAT-tailed polypeptides.
December 1, 2017: Trends in Cell Biology
https://www.readbyqxmd.com/read/29186125/promoter-bound-mettl3-maintains-myeloid-leukaemia-by-m6a-dependent-translation-control
#5
Isaia Barbieri, Konstantinos Tzelepis, Luca Pandolfini, Junwei Shi, Gonzalo Millán-Zambrano, Samuel C Robson, Demetrios Aspris, Valentina Migliori, Andrew J Bannister, Namshik Han, Etienne De Braekeleer, Hannes Ponstingl, Alan Hendrick, Christopher R Vakoc, George S Vassiliou, Tony Kouzarides
N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice...
November 27, 2017: Nature
https://www.readbyqxmd.com/read/29158335/a-deconvolution-protocol-for-chip-seq-reveals-analogous-enhancer-structures-on-the-mouse-and-human-ribosomal-rna-genes
#6
Jean-Clement Mars, Marianne Sabourin-Felix, Michel G Tremblay, Tom Moss
The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein-DNA interaction profiles. Here we describe a simple numerical deconvolution approach that in large part corrects for this variability and significantly improves both the resolution and quantitation of protein-DNA interaction maps deduced from ChIP-Seq data...
November 20, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29155943/logical-engineering-of-d-arm-and-t-stem-of-trna-that-enhances-d-amino-acid-incorporation
#7
Takayuki Katoh, Yoshihiko Iwane, Hiroaki Suga
A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA's D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid...
November 16, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29147007/modulating-cellular-balance-of-rps3-mono-ubiquitination-by-both-hel2-e3-ligase-and-ubp3-deubiquitinase-regulates-protein-quality-control
#8
Youjin Jung, Hag Dong Kim, Hee Woong Yang, Hye Jin Kim, Chang-Young Jang, Joon Kim
When a ribosome complex is stalled during the translation elongation process in eukaryotes, the mono-ubiquitination of Rps3 has recently been shown to be critical to ribosome quality control. We have discovered that the regulatory role of Rps3 mono-ubiquitination is controlled by a deubiquitinase. We also showed that an autophagic signal appears to be coupled to the mono-ubiquitination of Rps3p through the entrance of Ubp3p into the autophagosome in yeasts. The mono-ubiquitination of the Rps3 protein is tightly modulated by reciprocal action between the Hel2p E3 ligase and the Ubp3p deubiquitinase in yeasts and the reciprocal action between the RNF123 E3 ligase and the USP10 deubiquitinase in mammalian cells...
November 17, 2017: Experimental & Molecular Medicine
https://www.readbyqxmd.com/read/29129743/comprehensive-identification-of-proteins-binding-to-rna-g-quadruplex-motifs-in-the-5-utr-of-tumor-associated-mrnas
#9
Tatsuo Serikawa, Christos Spanos, Annekathrin von Hacht, Nediljko Budisa, Juri Rappsilber, Jens Kurreck
G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry...
November 9, 2017: Biochimie
https://www.readbyqxmd.com/read/29112848/vms1-relieves-a-mitochondrial-import-chokehold
#10
Lilian Lamech, Cole M Haynes
Ribosome stalling results in the production of truncated proteins that can cause proteotoxic stress if not efficiently degraded. A recent paper by Izawa et al. (2017) identifies Vms1 as a key player in the regulation of ribosome quality control specifically on mitochondria-localized ribosomes, ultimately preventing protein aggregate accumulation within mitochondria.
November 6, 2017: Developmental Cell
https://www.readbyqxmd.com/read/29107329/cytosolic-protein-vms1-links-ribosome-quality-control-to-mitochondrial-and-cellular-homeostasis
#11
Toshiaki Izawa, Sae-Hun Park, Liang Zhao, F Ulrich Hartl, Walter Neupert
Eukaryotic cells have evolved extensive protein quality-control mechanisms to remove faulty translation products. Here, we show that yeast cells continually produce faulty mitochondrial polypeptides that stall on the ribosome during translation but are imported into the mitochondria. The cytosolic protein Vms1, together with the E3 ligase Ltn1, protects against the mitochondrial toxicity of these proteins and maintains cell viability under respiratory conditions. In the absence of these factors, stalled polypeptides aggregate after import and sequester critical mitochondrial chaperone and translation machinery...
October 24, 2017: Cell
https://www.readbyqxmd.com/read/29100052/structural-basis-for-polyproline-mediated-ribosome-stalling-and-rescue-by-the-translation-elongation-factor-ef-p
#12
Paul Huter, Stefan Arenz, Lars V Bock, Michael Graf, Jan Ole Frister, Andre Heuer, Lauri Peil, Agata L Starosta, Ingo Wohlgemuth, Frank Peske, Jiří Nováček, Otto Berninghausen, Helmut Grubmüller, Tanel Tenson, Roland Beckmann, Marina V Rodnina, Andrea C Vaiana, Daniel N Wilson
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P...
November 2, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29097228/involvement-of-ppid-in-sec-dependent-protein-translocation
#13
Michaela Fürst, Yufan Zhou, Jana Merfort, Matthias Müller
The periplasmic space in between the inner and outer membrane of Gram-negative bacteria contains numerous chaperones that are involved in the biogenesis and rescue of extra-cytosolic proteins. In contrast to most of those periplasmic chaperones, PpiD is anchored by an N-terminal transmembrane domain within the inner membrane of Escherichia coli. There it is located in close proximity to the SecY subunit of the SecYEG translocon, which is the primary transporter for secretory and membrane proteins. By site-specific cross-linking we now found the periplasmic domain of PpiD also in close vicinity to the SecG subunit of the Sec translocon and we provide the first direct evidence for a functional cooperation between PpiD and the Sec translocon...
October 31, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29096076/regulation-of-mrna-translation-in-neurons-a-matter-of-life-and-death
#14
REVIEW
Mridu Kapur, Caitlin E Monaghan, Susan L Ackerman
Dynamic regulation of mRNA translation initiation and elongation is essential for the survival and function of neural cells. Global reductions in translation initiation resulting from mutations in the translational machinery or inappropriate activation of the integrated stress response may contribute to pathogenesis in a subset of neurodegenerative disorders. Aberrant proteins generated by non-canonical translation initiation may be a factor in the neuron death observed in the nucleotide repeat expansion diseases...
November 1, 2017: Neuron
https://www.readbyqxmd.com/read/29082557/the-nuclear-dek-interactome-supports-multi-functionality
#15
Eric A Smith, Eric F Krumpelbeck, Anil G Jegga, Malte Prell, Marie M Matrka, Ferdinand Kappes, Kenneth D Greis, Abdullah M Ali, Amom R Meetei, Susanne I Wells
DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry...
October 30, 2017: Proteins
https://www.readbyqxmd.com/read/29069457/nol12-is-a-multifunctional-rna-binding-protein-at-the-nexus-of-rna-and-dna-metabolism
#16
Daniel D Scott, Christian Trahan, Pierre J Zindy, Lisbeth C Aguilar, Marc Y Delubac, Eric L Van Nostrand, Srivathsan Adivarahan, Karen E Wei, Gene W Yeo, Daniel Zenklusen, Marlene Oeffinger
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm...
October 23, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29034854/whole-genome-transcriptomics-reveals-global-effects-including-up-regulation-of-francisella-pathogenicity-island-gene-expression-during-active-stringent-response-in-the-highly-virulent-francisella-tularensis-subsp-tularensis-schu-s4
#17
Amber L Murch, Paul J Skipp, Peter L Roach, Petra C F Oyston
During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression...
October 16, 2017: Microbiology
https://www.readbyqxmd.com/read/28977579/the-rio1p-atpase-hinders-premature-entry-into-translation-of-late-pre-40s-pre-ribosomal-particles
#18
Kamila Belhabich-Baumas, Clément Joret, Beáta E Jády, Célia Plisson-Chastang, Ramtin Shayan, Christophe Klopp, Anthony K Henras, Yves Henry, Annie Mougin
Cytoplasmic maturation of precursors to the small ribosomal subunit in yeast requires the intervention of a dozen assembly factors (AFs), the precise roles of which remain elusive. One of these is Rio1p that seems to intervene at a late step of pre-40S particle maturation. We have investigated the role played by Rio1p in the dynamic association and dissociation of AFs with and from pre-40S particles. Our results indicate that Rio1p depletion leads to the stalling of at least 4 AFs (Nob1p, Tsr1p, Pno1p/Dim2p and Fap7p) in 80S-like particles...
October 13, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28973849/miscoding-induced-stalling-of-substrate-translocation-on-the-bacterial-ribosome
#19
Jose L Alejo, Scott C Blanchard
Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex...
October 10, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28973451/the-abundance-of-fob1-modulates-the-efficiency-of-rrfbs-to-stall-replication-forks
#20
Alicia Castán, Pablo Hernández, Dora B Krimer, Jorge B Schvartzman
In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3' end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1...
September 29, 2017: Nucleic Acids Research
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