Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.This work was supported by grants from Science Foundation Ireland [12/IP/1492 and 13/1A/1853 to J.F.A; 12/IA/1335 to P.V.B.], US. National Institutes of Health [RO3 MH098688 to J.F.A.], the Wellcome Trust [106207 to A.E.F and 094423 to P.V.B.] and the European Research Council (ERC) grant No. 646891 to A.E.F.]This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/nar/gkw53

Towards an In Vitro Reconstitution of the Alpha-Carboxysome

Many bacteria employ a protein organelle, the carboxysome, to concentrate carbon dioxideand catalyze the initial fixation reaction. Only 10 genes from Halothiobacillusneapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli,opening the door to mechanistic analysis of the assembly process of this 200 MDa+complex. One of these genes, csoS2, produces two highly repetitive intrinsically-disorderedprotein isoforms and has been shown to be indispensable in carboxysome assembly.Detailed functional characterization of csoS2, however, has been hindered by the lack ofunderstanding of how this single gene yields expression of two gene products. In this work,we set out to develop a deeper understanding of CsoS2's biogenesis and its function in α-carboxysome assembly. Using tandem mass spectrometry and biochemical assays, we haverevealed that -1 programmed ribosomal frameshifting (- 1 PRF) is responsible for thegeneration of a truncated protein with C-terminus translated from the -1 frame, CsoS2A, inaddition to the full-length protein, CsoS2B. We show for the first time that CsoS2B can beindependently produced by mutations of -1 PRF elements and only CsoS2B is necessary forthe assembly of H. neapolitanus carboxysomes in native and heterologous hosts. With theknowledge of the identity of CsoS2 isoforms, we next investigate the ability of individualCsoS2 domains to participate in protein-protein interaction with RuBisCO, the primaryenzymatic component of the carboxysome. Here, we demonstrate that the 259-aa N-terminal domain of CsoS2 multivalently binds RuBisCO, potentially via its shortamphiphatic helices. Finally, based on our findings, we propose a hypothetical model thatdescribes the formation and maturation of the α-carboxysome. This work illustrates, forthe first time, the simultaneous involvement of cotranslational regulation and anintrinsically-disordered protein in the assembly of a prokaryotic organelle

From the basics to emerging diagnostic technologies: What is on the horizon for tilapia disease diagnostics?

Tilapia is an affordable protein source farmed in over 140 countries with the majority of production in low- and middle-income countries. Intensification of tilapia farming has exacerbated losses caused by emerging and re-emerging infectious diseases. Disease diagnostics play a crucial role in biosecurity and health management to mitigate disease loss and improve animal welfare in aquaculture. Three continuous levels of diagnostics (I, II and III) for aquatic species have been proposed by Food and Agriculture Organization of the United Nations (FAO) and the Network of Aquaculture Centers in Asia and the Pacific (NACA) to promote the integration of basic and advanced methods to achieve accurate and meaningful interpretation of diagnostic results. However, the recent proliferation of cutting-edge molecular methods applied in the diagnosis of diseases of aquacultured animals has shifted the focus of researchers and users away from basic approaches and toward molecular diagnostics, despite the fact that many diseases can be rapidly diagnosed using inexpensive, simple microscopic examination and that most emerging diseases in aquaculture were discovered by histopathology. This review, therefore, revisits and highlights the importance of the three levels of diagnostics for diseases of tilapia, particularly the frequently overlooked basic procedures (e.g., case history records, gross pathology, presumptive diagnostic methods and histopathology). The review also covers current and emerging molecular diagnostic technologies for tilapia pathogens including polymerase chain reaction methods (conventional, quantitative, digital), isothermal amplification methods Loop-mediated Isothermal Amplification (LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)-based detection, lateral flow immunoassays, as well as discussing what is on the horizon for tilapia disease diagnostics (next generation sequencing, artificial intelligence, environmental Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) and point-of-care testing) providing a future vision for transferring these technologies to farmers and stakeholders for a sustainable aquatic food system transformation