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

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.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Bi-directional ribosome scanning controls the stringency of start codon selection

The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5ʹ→3ʹ migration of the pre-initiation complex (PIC) along the 5ʹ UTR. In probing translation initiation from ultra-short 5ʹ UTR, we report that an AUG triplet near the 5ʹ end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5ʹ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5ʹ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.

Biosynthesis and Degradation of Sulfur Modifications in tRNAs

Various sulfur-containing biomolecules include iron–sulfur clusters that act as cofactors for enzymes, sulfur-containing vitamins such as thiamin, and sulfur-modified nucleosides in RNA, in addition to methionine and cysteine in proteins. Sulfur-containing nucleosides are post-transcriptionally introduced into tRNA molecules, where they ensure precise codon recognition or stabilization of tRNA structure, thereby maintaining cellular proteome integrity. Modulating sulfur modification controls the translation efficiency of specific groups of genes, allowing organisms to adapt to specific environments. The biosynthesis of tRNA sulfur nucleosides involves elaborate ‘sulfur trafficking systems’ within cellular sulfur metabolism and ‘modification enzymes’ that incorporate sulfur atoms into tRNA. This review provides an up-to-date overview of advances in our knowledge of the mechanisms involved. It covers the functions, biosynthesis, and biodegradation of sulfur-containing nucleosides as well as the reaction mechanisms of biosynthetic enzymes catalyzed by the iron–sulfur clusters, and identification of enzymes involved in the de-modification of sulfur atoms of RNA. The mechanistic similarity of these opposite reactions is discussed. Mutations in genes related to these pathways can cause human diseases (e.g., cancer, diabetes, and mitochondrial diseases), emphasizing the importance of these pathways.

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

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.

Inosine in Biology and Disease

The nucleoside inosine plays an important role in purine biosynthesis, gene translation, and modulation of the fate of RNAs. The editing of adenosine to inosine is a widespread post-transcriptional modification in transfer RNAs (tRNAs) and messenger RNAs (mRNAs). At the wobble position of tRNA anticodons, inosine profoundly modifies codon recognition, while in mRNA, inosines can modify the sequence of the translated polypeptide or modulate the stability, localization, and splicing of transcripts. Inosine is also found in non-coding and exogenous RNAs, where it plays key structural and functional roles. In addition, molecular inosine is an important secondary metabolite in purine metabolism that also acts as a molecular messenger in cell signaling pathways. Here, we review the functional roles of inosine in biology and their connections to human health.

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

How Many Messenger RNAs Can Be Translated by the START Mechanism?

Translation initiation is a key step in the protein synthesis stage of the gene expression pathway of all living cells. In this important process, ribosomes have to accurately find the AUG start codon in order to ensure the integrity of the proteome. “Structure Assisted RNA Translation”, or “START”, has been proposed to use stable secondary structures located in the coding sequence to augment start site selection by steric hindrance of the progression of pre-initiation complex on messenger RNA. This implies that such structures have to be located downstream and at on optimal distance from the AUG start codon (i.e., downstream nucleotide +16). In order to assess the importance of the START mechanism in the overall mRNA translation process, we developed a bioinformatic tool to screen coding sequences for such stable structures in a 50 nucleotide-long window spanning the nucleotides from +16 to +65. We screened eight bacterial genomes and six eukaryotic genomes. We found stable structures in 0.6–2.5% of eukaryotic coding sequences. Among these, approximately half of them were structures predicted to form G-quadruplex structures. In humans, we selected 747 structures. In bacteria, the coding sequences from Gram-positive bacteria contained 2.6–4.2% stable structures, whereas the structures were less abundant in Gram-negative bacteria (0.2–2.7%). In contrast to eukaryotes, putative G-quadruplex structures are very rare in the coding sequence of bacteria. Altogether, our study reveals that the START mechanism seems to be an ancient strategy to facilitate the start codon recognition that is used in different kingdoms of life.