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Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

ABSTRACT: The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete Didymium to replace the mRNA m⁷G cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2', 5' phosphodiester bond. We show that the ribozyme functions in vivo in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m⁷G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m⁷G-capped counterpart, consistent with a key role for the m⁷G cap in mRNA turnover. Our study emphasizes important activities of the m⁷G cap and suggests new utilities of lariat capping as a molecular tool in vivo.

SUBMITTER: Krogh N

PROVIDER: S-EPMC5393178 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

Krogh Nicolai N Pietschmann Max M Schmid Manfred M Jensen Torben Heick TH Nielsen Henrik H

RNA (New York, N.Y.) 20170203 5

The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete <i>Didymium</i> to replace the mRNA m<sup>7</sup>G cap of a single reporter mRNA species with a tiny lariat in which the first and ...[more]

PMID: 28159804

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Project description:Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

Dataset Information

Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

Publications

Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

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