scholarly journals Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

2019 ◽  
Author(s):  
Jonathan Bohlen ◽  
Kai Fenzl ◽  
Günter Kramer ◽  
Bernd Bukau ◽  
Aurelio A. Teleman
Keyword(s):  
Translation Initiation ◽  
Human Cells ◽  
Open Reading Frames ◽  
Translation Regulation ◽  
List Type ◽  
Translation Efficiency ◽  
Initiation Factors ◽  
Upstream Open Reading Frames

SUMMARYTranslation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ∼12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.HIGHLIGHTSSelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivoScanning ribosomes are cap-tethered in human cellsOnly one ribosome scans a 5’UTR at a time in human cellsRibosomes retain eIFs during early translation, allowing reinitiation after uORFs

scholarly journals Near-Cognate Codons Contribute Complexity to Translation Regulation

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
N. Louise Glass
Keyword(s):  
Protein Synthesis ◽  
Cell Fate ◽  
Translation Initiation ◽  
Cellular Response ◽  
Open Reading Frames ◽  
Translation Regulation ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Upstream Open Reading Frames ◽  
Response To Stress

ABSTRACT The interplay between translation initiation, modification of translation initiation factors, and selection of start sites on mRNA for protein synthesis can play a regulatory role in the cellular response to stress, development, and cell fate in eukaryotic species by shaping the proteome. As shown by Ivanov et al. (mBio 8:e00844-17, 2017, https://doi.org/10.1128/mBio.00844-17 !), in the filamentous fungus Neurospora crassa, both upstream open reading frames (uORFs) and near-cognate start codons negatively or positively regulate the translation of the transcription factor CPC1 and production of CPC1 isoforms, which mediate the cellular response to amino acid starvation. Dissecting the physiological roles that differentiate cellular choice of translation initiation is an important parameter to understanding mechanisms that determine cell fate via gene regulation and protein synthesis.

scholarly journals Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation

2007 ◽  
Vol 283 (3) ◽  
pp. 1340-1349 ◽  
Author(s):  
Mateen A. Khan ◽  
Hiroshi Miyoshi ◽  
Daniel R. Gallie ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Wheat Germ ◽  
Mrna Translation ◽  
Kinetic Studies ◽  
Dissociation Rate ◽  
In Vitro Translation ◽  
Translation Efficiency ◽  
Viral Mrna ◽  
Initiation Factors

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show ∼2.6-fold faster association for eIFiso4F·VPg as compared with eIF4F·VPg. The dissociation rate was ∼2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F·VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.

scholarly journals The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jyothsna Visweswaraiah ◽  
Yvette Pittman ◽  
Thomas E Dever ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Exit Channel ◽  
Hairpin Loop ◽  
Leaky Scanning ◽  
Initiation Factors ◽  
Codon Recognition

The eukaryotic 43S pre-initiation complex bearing tRNAiMet scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred ‘leaky-scanning’ of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the ‘PIN’ conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.

scholarly journals Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

2000 ◽  
Vol 20 (19) ◽  
pp. 7183-7191 ◽  
Author(s):  
Sang Ki Choi ◽  
DeAnne S. Olsen ◽  
Antonina Roll-Mecak ◽  
Agnes Martung ◽  
Keith L. Remo ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Sequence Similarity ◽  
Start Codon ◽  
Amino Acid Sequence Similarity ◽  
Physical Interaction ◽  
Binding Assays ◽  
Initiation Factors ◽  
Translation Initiation Factors

ABSTRACT To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

scholarly journals eIF4G-driven translation initiation of downstream ORFs in mammalian cells

2020 ◽  
Vol 48 (18) ◽  
pp. 10441-10455
Author(s):  
Risa Nobuta ◽  
Kodai Machida ◽  
Misaki Sato ◽  
Satoshi Hashimoto ◽  
Yasuhito Toriumi ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Translation System ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Genome Wide

Abstract Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3′ untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3′ UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3′ UTRs. The 3′ UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3′ UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3′ UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3′ UTRs of mRNAs may encode various products.

scholarly journals Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

2002 ◽  
Vol 76 (5) ◽  
pp. 2062-2074 ◽  
Author(s):  
N. Muge Kuyumcu-Martinez ◽  
Michelle Joachims ◽  
Richard E. Lloyd
Keyword(s):  
Translation Initiation ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
3C Protease

ABSTRACT Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases.

scholarly journals Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 323
Author(s):  
Piotr Gawroński ◽  
Aleksandra Pałac ◽  
Lars B. Scharff
Keyword(s):  
Gene Expression ◽  
Secondary Structure ◽  
Translation Initiation ◽  
Structural Information ◽  
Regulation Of Gene Expression ◽  
Dimethyl Sulfate ◽  
Translation Efficiency ◽  
Mrna Secondary Structure

mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

scholarly journals Operon mRNAs are organized into ORF-centric structures that predict translation efficiency

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
David H Burkhardt ◽  
Silvi Rouskin ◽  
Yan Zhang ◽  
Gene-Wei Li ◽  
Jonathan S Weissman ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Open Reading Frames ◽  
Reduced Form ◽  
Translation Efficiency ◽  
Translation Rate ◽  
Polycistronic Mrna ◽  
Secondary Structure Analysis ◽  
Mrna Structure

Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF.

scholarly journals Dendritic BC1 RNA in translational control mechanisms

2005 ◽  
Vol 171 (5) ◽  
pp. 811-821 ◽  
Author(s):  
Huidong Wang ◽  
Anna Iacoangeli ◽  
Daisy Lin ◽  
Keith Williams ◽  
Robert B. Denman ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translational Control ◽  
Fragile X ◽  
Initiation Factor ◽  
Control Mechanisms ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Specific Effector

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

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