Angiogenin-induced tRNA fragments inhibit translation initiation

Angiogenin is a stress-activated ribonuclease that cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs). Transfection of natural or synthetic tiRNAs inhibits protein synthesis and triggers the phospho-eIF2α-independent assembly of stress granules (SGs), essential components of the stress response program. We show that selected tiRNAs inhibit protein synthesis by displacing eIF4G/eIF4A from uncapped > capped RNAs. tiRNAs also displace eIF4F, but not eIF4E:4EBP1, from isolated m(7)G cap. We identify a terminal oligoguanine motif that is required to displace the eIF4F complex, inhibit translation, and?induce SG assembly. We show that the tiRNA-associated translational silencer YB-1 contributes to angiogenin-, tiRNA-, and oxidative stress-induced translational repression. Our data reveal some of the mechanisms by which stress-induced tRNA cleavage inhibits protein synthesis and activates a cytoprotective stress response program.     

Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNA^Met synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNA^Met and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 ‘writes’ modifications on tRNA and mRNA, and both types of RNAs are ‘read’ by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.     

mRNA翻译起始区二级结构改变对干扰素基因在大肠杆菌中翻译的影响

为研究mRNA翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变α8干扰素及αA干扰素衍生物基因的5′端若干位点,使其与表达载体重组后转录形成的mRNA翻译起始区结构发生改变。SDS-PAGE及活性测定证实这些改变提高了外源基因的表达水平。RNA斑点印迹表明突变前后基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高。mRNA翻译起始区二级结构预测提示其生成自由能(ΔG)的变化可能与表达水平的提高有关。     

Unfolding of mRNA secondary structure by the bacterial translation initiation complex

Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.     

Comparison of mRNA features affecting translation initiation and reinitiation

Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.     

Eukaryotic and archaeal translation initiation factor 2: A heterotrimeric tRNA carrier

Eukaryotic/archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that plays a key role in selection of the correct start codon on messenger RNA. This review integrates structural and functional data to discuss the involvement of the three subunits in initiator tRNA binding. A possible role of the peripheral subunits in modulating the guanine nucleotide cycle on the core subunit is also addressed.     

Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency

Precise prediction of prokaryotic translation efficiency can provide valuable information for optimizing bacterial host for the production of biochemical compounds or recombinant proteins. However, dynamic changes in mRNA folding throughout translation make it difficult to assess translation efficiency. Here, we systematically determined the universal folding regions that significantly affect the efficiency of translation in Escherichia coli. By assessing the specific regions for mRNA folding, we could construct a predictive design method, UTR Designer, and demonstrate that proper codon optimization around the 5′-proximal coding sequence is necessary to achieve a broad range of expression levels. Finally, we applied our method to control the threshold value of input signals switching on a genetic circuit. This should increase our understanding of the processes underlying gene expression and provide an efficient design principle for optimizing various biological systems, thereby facilitating future efforts in metabolic engineering and synthetic biology.     

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.     

Signaling pathways that control mRNA translation initiation in macrophages

Translational control in mammalian cells plays a critical role in regulating differentiation, cell growth, cell cycle and response to diverse stresses. Macrophages are one of the most versatile cell types in the body. They are professional phagocytic cells that can be found in almost all tissues and adapt tissue-specific functions. Recent studies highlight the importance of translational control in macrophages during invasion of pathogens, exposure to cytokines and in the context of tissue specific functions. In this review, we summarize the current knowledge regarding the role of mRNA translational control in regulation of macrophages.     

Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.     

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

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Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.