scholarly journals eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

2022 ◽  
Vol 8 ◽  
Author(s):  
Andrei Stanciu ◽  
Juncheng Luo ◽  
Lucy Funes ◽  
Shanya Galbokke Hewage ◽  
Colin Echeverría Aitken
Keyword(s):  
Translation Initiation ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Start Codon ◽  
Untranslated Regions ◽  
Initiation Factors ◽  
Codon Recognition ◽  
Entire Complex ◽  
The Individual

Translation initiation in eukaryotes is a multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions—and disentangling the roles of the individual subunits of the eIF3 complex—remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome and which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5′-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5′-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

scholarly journals Fidelity of translation initiation is required for coordinated respiratory complex assembly

2019 ◽  
Vol 5 (12) ◽  
pp. eaay2118 ◽  
Author(s):  
Danielle L. Rudler ◽  
Laetitia A. Hughes ◽  
Kara L. Perks ◽  
Tara R. Richman ◽  
Irina Kuznetsova ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mitochondrial Protein ◽  
Molecular Machines ◽  
Ribosome Profiling ◽  
Untranslated Regions ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Initiation Factors ◽  
Mitochondrial Ribosomes

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle–specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

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 Eukaryotic aspects of translation initiation brought into focus

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160186 ◽  
Author(s):  
Christopher H. S. Aylett ◽  
Nenad Ban
Keyword(s):  
Translation Initiation ◽  
Structural Information ◽  
Start Codon ◽  
Translation Regulation ◽  
Initiation Factors ◽  
Codon Recognition ◽  
Initiation Of Translation ◽  
Control Translation ◽  
Additional Level ◽  
Subunit Joining

In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

Genetics ◽  
2021 ◽  
Author(s):  
Jinsheng Dong ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Yeast Mutant ◽  
Initiation Complex ◽  
Initiation Factors ◽  
Codon Recognition ◽  
Open Conformation ◽  
Kozak Context

Abstract The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that non-lethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

scholarly journals Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

2019 ◽  
Author(s):  
Susan Wagner ◽  
Anna Herrmannová ◽  
Vladislava Hronová ◽  
Neelam Sen ◽  
Ross D. Hannan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Mammalian Cells ◽  
Regulation Of Gene Expression ◽  
Ribosome Profiling ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
Initiation Phase ◽  
Gene Expression Studies

SUMMARYTranslational control targeting mainly the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast Translational Complex Profile sequencing (TCP-seq), related to ribosome profiling, and adopted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ses) in addition to 80S ribosomes (80Ses), revealed that mammalian and yeast 40Ses distribute similarly across 5’UTRs indicating considerable evolutionary conservation. We further developed a variation called Selective TCP-seq (Sel-TCP-seq) enabling selection for 40Ses and 80Ses associated with an immuno-targeted factor in yeast and human. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5’UTRs with scanning 40Ses to successively dissociate upon start codon recognition. Manifesting the Sel-TCP-seq versatility for gene expression studies, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

scholarly journals Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

2019 ◽  
Vol 20 (18) ◽  
pp. 4464 ◽  
Author(s):  
Nikolay E. Shirokikh ◽  
Yulia S. Dutikova ◽  
Maria A. Staroverova ◽  
Ross D. Hannan ◽  
Thomas Preiss
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Primer Extension ◽  
Initiation Factor ◽  
Start Codon ◽  
Untranslated Regions ◽  
Specific Rate ◽  
Messenger Ribonucleic Acid ◽  
Free Translation

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA (‘toeprinting’), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This ‘−8 nt toeprint’ was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the ‘−8 nt toeprint’ was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.

scholarly journals Translation Initiation Factor 2γ Mutant Alters Start Codon Selection Independent of Met-tRNA Binding

2008 ◽  
Vol 28 (22) ◽  
pp. 6877-6888 ◽  
Author(s):  
Pankaj V. Alone ◽  
Chune Cao ◽  
Thomas E. Dever
Keyword(s):  
Binding Affinity ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
40S Ribosomal Subunit ◽  
Initiation Factor 2

ABSTRACT Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNAi Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNAi Met to the 40S ribosomal subunit, and previous studies identified Sui− mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNAi Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNAi Met binding and causes a Sui− phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNAi Met binding affinity and cell growth; however, only A208V suppresses the Sui− phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNAi Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui− phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui− phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNAi Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAi Met binding affinity.

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