Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.

/pdf/genome-wide-analysis-in-vivo-of-translation-with-nucleotide-36vshepmua.pdf

Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

https://escholarship.org/content/qt56q617w2/qt56q617w2.pdf?t=qblgcg

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.

Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the recently identified Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV).

/pdf/coronaviruses-an-overview-of-their-replication-and-4lyvr9vdqs.pdf

Coronaviruses: An Overview of Their Replication and Pathogenesis

Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes

Natural products have been a rich source of compounds for drug discovery. However, their use has diminished in the past two decades, in part because of technical barriers to screening natural products in high-throughput assays against molecular targets. Here, we review strategies for natural product screening that harness the recent technical advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.

/pdf/the-re-emergence-of-natural-products-for-drug-discovery-in-1h0jebzmkp.pdf

The re-emergence of natural products for drug discovery in the genomics era

Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot.

Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5′ cap structure or the presence of highly structured 5′UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases.

Non-canonical translation in RNA viruses.

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.

/pdf/a-mechanical-explanation-of-rna-pseudoknot-function-in-115kzfcjnv.pdf

A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting

The polymerase-encoding region of the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains two very large, briefly overlapping open reading frames (ORF), F1 and F2, and it has been suggested on the basis of sequence analysis that expression of the downstream ORF, F2, might be mediated through ribosomal frame-shifting. To examine this possibility a cDNA fragment containing the F1/F2 overlap region was cloned within a marker gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Messenger RNA transcribed from this plasmid, when translated in cell-free systems, specified the synthesis of polypeptides whose size was entirely consistent with the products predicted by an efficient ribosomal frame-shifting event within the overlap region. The nature of the products was confirmed by their reactivity with antisera raised against defined portions of the flanking marker gene. This is the first non-retroviral example of ribosomal frame-shifting in higher eukaryotes.

An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV.

https://www.cell.com/molecular-cell/pdf/S1097-2765(04)00031-0.pdf

Ian Brierley

Papers

Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot.

Non-canonical translation in RNA viruses.

A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting

An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV.

Reprogrammed genetic decoding in cellular gene expression