denver-papillae-protocol-for-objective-analysis-of-fungiform-papillae

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Denver Museum of Nature & Science

How viral transcripts are protected from the cellular RNA decay machinery and the importance of this protection for the virus are largely unknown. We demonstrate that Sindbis virus, a prototypical single-stranded arthropod-borne alphavirus, uses U-rich 3' UTR sequences in its RNAs to recruit a known regulator of cellular mRNA stability, the HuR protein, during infections of both human and vector mosquito cells. HuR binds viral RNAs with high specificity and affinity. Sindbis virus infection induces the selective movement of HuR out of the mammalian cell nucleus, thereby increasing the available cytoplasmic HuR pool. Finally, knockdown of HuR results in a significant increase in the rate of decay of Sindbis virus RNAs and diminishes viral yields in both human and mosquito cells. These data indicate that Sindbis virus and likely other alphaviruses usurp the HuR protein to avoid the cellular mRNA decay machinery and maintain a highly productive infection.

Sindbis virus usurps the cellular HuR protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells.

When considering the control of gene expression, the focus has traditionally been on transcriptional regulation. Recently, however, the large contribution made by mRNA decay has become difficult to ignore. Large-scale analyses indicate that as many as half of all changes in the amounts of mRNA in some responses can be attributed to altered rates of decay. In this article, we discuss some of the mechanisms that are used by the cell to mediate and regulate this intriguing process.

The highways and byways of mRNA decay.

The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5' cap and a 3' poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3' untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3' UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors-including a 38-kDa protein-were implicated in the repression of deadenylation mediated by the SINV 3' UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3' UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.

The 3' untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and Mammalian cells.

The field of RNA decay has grown extensively over the last few years and numerous decay pathways have been identified and characterized. This is a truly powerful machinery for both regulation and quality control of gene expression. It is very likely that the transcripts of RNA viruses must successfully confront this arsenal of enzymes and RNA binding factors in order to establish a productive infection. This interface is an understudied branch of virology that needs to be explored if we are to fully comprehend the molecular biology of virus-cell interactions. Research in this area has the potential to increase our understanding of the fundamentals of both mRNA stability and viral biology, perhaps leading to novel antiviral approaches. This chapter discusses methods for examining the half-lives of viral RNAs during natural infection, including purification of the viral transcripts and subsequent analysis of both deadenylation and decay. Additionally, a hybrid selection protocol for identifying viral-specific small RNAs that are generated during infection by the RNAi branch of the cellular RNA decay machinery is described.

Chapter 5. In vivo analysis of the decay of transcripts generated by cytoplasmic RNA viruses.

Understanding the influence of taste perception on food choice has captured the interest of academics, industry, and the general public, the latter as evidenced by the extent of popular media coverage and use of the term supertaster. Supertasters are highly sensitive to the bitter tastant propylthiouracil (PROP) and its chemical relative phenylthiocarbamide. The well-researched differences in taste sensitivity to these bitter chemicals are partially controlled by variation in the TAS2R38 gene; however, this variation alone does not explain the supertaster phenomenon. It has been suggested that density of papillae, which house taste buds, may explain supertasting. To address the unresolved role of papillae, we used crowdsourcing in the museum-based Genetics of Taste Lab. This community lab is uniquely situated to attract both a large population of human subjects and host a team of citizen scientists to research population-based questions about human genetics, taste, and health. Using this model, we find that PROP bitterness is not in any way predicted by papillae density. This result holds within the whole sample, when divided into major diplotypes, and when correcting for age, sex, and genotype. Furthermore, it holds when dividing participants into oft-used taster status groups. These data argue against the use of papillae density in predicting taste sensitivity and caution against imprecise use of the term supertaster. Furthermore, it supports a growing volume of evidence that sets the stage for hypergeusia, a reconceptualization of heightened oral sensitivity that is not based solely on PROP or papillae density. Finally, our model demonstrates how community-based research can serve as a unique venue for both study participation and citizen science that makes scientific research accessible and relevant to people's everyday lives.

Article	Year
Sindbis virus usurps the cellular HuR protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells. Cell host & microbe\| PubMed ID: 20709296	2010
The highways and byways of mRNA decay. Nature reviews. Molecular cell biology\| PubMed ID: 17245413	2007
The 3' untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and Mammalian cells. Journal of virology\| PubMed ID: 17977976	2008
Chapter 5. In vivo analysis of the decay of transcripts generated by cytoplasmic RNA viruses. Methods in enzymology\| PubMed ID: 19215755	2008
Crowdsourcing taste research: genetic and phenotypic predictors of bitter taste perception as a model. Frontiers in integrative neuroscience\| PubMed ID: 24904324	2014

Nicole L. Garneau

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