Yeast p20 is a small, acidic protein that binds eIF4E, the cap-binding protein. It has been proposed to affect mRNA translation and degradation, however p20's function as an eIF4E-binding protein ...(4E-BP) and its physiological significance has not been clearly established. In this paper we present data demonstrating that p20 is capable of binding directly to mRNA due to electrostatic interaction of a stretch of arginine and histidine residues in the protein with negatively charged phosphates in the mRNA backbone. This interaction contributes to formation of a ternary eIF4E/p20/capped mRNA complex that is more stable than complexes composed of capped mRNA bound to eIF4E in the absence of p20. eIF4E/p20 complex was found to have a more pronounced stimulatory effect on capped mRNA translation than purified eIF4E alone. Addition of peptides containing the eIF4E-binding domains present in p20 (motif YTIDELF), in eIF4G (motif YGPTFLL) or Eap1 (motif YSMNELY) completely inhibited eIF4E-dependent capped mRNA translation (in vitro), but had a greatly reduced inhibitory effect when eIF4E/p20 complex was present. We propose that the eIF4E/p20/mRNA complex serves as a stable depository of mRNAs existing in a dynamic equilibrium with other complexes such as eIF4E/eIF4G (required for translation) and eIF4E/Eap1 (required for mRNA degradation).
GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports ...implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNA
, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.
Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, ...comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses.
Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence.
We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant.
Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost ...every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion.
•Key methods available for measuring co-translational protein folding are described.•Emphasis is placed on modern, real-time approaches.•Comprehensive information on nascent polypeptide chains ...folding is presented.
Advances in techniques such as nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and single-molecule and time-resolved fluorescent approaches are transforming our ability to study co-translational protein folding both in vivo in living cells and in vitro in reconstituted cell-free translation systems. These approaches provide comprehensive information on the spatial organization and dynamics of nascent polypeptide chains and the kinetics of co-translational protein folding. This information has led to an improved understanding of the process of protein folding in living cells and should allow remaining key questions in the field, such as what structures are formed within nascent chains during protein synthesis and when, to be answered. Ultimately, studies using these techniques will facilitate development of a unified concept of protein folding, a process that is essential for proper cell function and organism viability. This review describes current methods for analysis of co-translational protein folding with an emphasis on some of the recently developed techniques that allow monitoring of co-translational protein folding in real-time.
The genetic code sets the correspondence between the sequence of a given nucleotide triplet in an mRNA molecule, called a codon, and the amino acid that is added to the growing polypeptide chain ...during protein synthesis. With four bases (A, G, U, and C), there are 64 possible triplet codons: 61 sense codons (encoding amino acids) and 3 nonsense codons (so-called, stop codons that define termination of translation). In most organisms, there are 20 common/standard amino acids used in protein synthesis; thus, the genetic code is redundant with most amino acids (with the exception of Met and Trp) are being encoded by more than one (synonymous) codon. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in mRNA suggested that the specific codon choice might have functional implications beyond coding for amino acid. Observation of nonequivalent use of codons in mRNAs implied a possibility of the existence of auxiliary information in the genetic code. Indeed, it has been found that genetic code contains several layers of such additional information and that synonymous codons are strategically placed within mRNAs to ensure a particular translation kinetics facilitating and fine-tuning co-translational protein folding in the cell via step-wise/sequential structuring of distinct regions of the polypeptide chain emerging from the ribosome at different points in time. This review summarizes key findings in the field that have identified the role of synonymous codons and their usage in protein folding in the cell.
Gene expression is highly variable across tissues of multi-cellular organisms, influencing the codon usage of the tissue-specific transcriptome. Cancer disrupts the gene expression pattern of healthy ...tissue resulting in altered codon usage preferences. The topic of codon usage changes as they relate to codon demand, and tRNA supply in cancer is of growing interest.
We analyzed transcriptome-weighted codon and codon pair usage based on The Cancer Genome Atlas (TCGA) RNA-seq data from 6427 solid tumor samples and 632 normal tissue samples. This dataset represents 32 cancer types affecting 11 distinct tissues. Our analysis focused on tissues that give rise to multiple solid tumor types and cancer types that are present in multiple tissues.
We identified distinct patterns of synonymous codon usage changes for different cancer types affecting the same tissue. For example, a substantial increase in GGT-glycine was observed in invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), and mixed invasive ductal and lobular carcinoma (IDLC) of the breast. Change in synonymous codon preference favoring GGT correlated with change in synonymous codon preference against GGC in IDC and IDLC, but not in ILC. Furthermore, we examined the codon usage changes between paired healthy/tumor tissue from the same patient. Using clinical data from TCGA, we conducted a survival analysis of patients based on the degree of change between healthy and tumor-specific codon usage, revealing an association between larger changes and increased mortality. We have also created a database that contains cancer-specific codon and codon pair usage data for cancer types derived from TCGA, which represents a comprehensive tool for codon-usage-oriented cancer research.
Based on data from TCGA, we have highlighted tumor type-specific signatures of codon and codon pair usage. Paired data revealed variable changes to codon usage patterns, which must be considered when designing personalized cancer treatments. The associated database, CancerCoCoPUTs, represents a comprehensive resource for codon and codon pair usage in cancer and is available at https://dnahive.fda.gov/review/cancercocoputs/ . These findings are important to understand the relationship between tRNA supply and codon demand in cancer states and could help guide the development of new cancer therapeutics.
Introduction
Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled ...insight into genotype–phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non‐synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half‐life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated.
Aim
To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease‐associated mutations in haemophilia.
Methods
Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease‐associated and synonymous vs. non‐synonymous point mutations in F8, F9 and a third X‐linked gene, DMD (dystrophin).
Results
In F8 and DMD, disease‐associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101–151 nucleotide fragment length appears to be a feasible range for structuring future studies.
Conclusion
mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease‐associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.
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