Investigating BMV RNA2 Translation Regulation

To study BMV RNA2 translation in the absence and presence of hTRPV4, WT (Saccharomyces cerevisiae Meyen ex E.C. Hansen (ATCC^® 201388^™) and ΔYvc1 (YSC1053 (ThermoFisherScientific) yeast strains were transformed with pB2NR3. The ΔYvc1 strain was additionally transformed with pBT3-STE-TRPV4 or an empty plasmid. Cells were grown in 2% galactose and harvested in mid-log phase. Total protein was extracted and analyzed by western blot as previously described^{66 (link)}. Total RNA was extracted and RNA2 and Act1 were analyzed by quantitative PCR (qPCR) using qScript XLT One-Step RT-qPCR ToughMix from Quanta Biosciences.

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Doñate-Macián P., Jungfleisch J., Pérez-Vilaró G., Rubio-Moscardo F., Perálvarez-Marín A., Diez J, & Valverde M.A. (2018). The TRPV4 channel links calcium influx to DDX3X activity and viral infectivity. Nature Communications, 9, 2307.

Publication 2018

qScript XLT One-Step RT-qPCR ToughMix

Cells Galactose Plasmid Protein Rt pcr Strain Trpv4 Western blot Yeast

Corresponding Organization :

Other organizations : Pompeu Fabra University, Universitat Autònoma de Barcelona

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Variable analysis

independent variables

Presence of hTRPV4 in the ΔYvc1 yeast strain

dependent variables

BMV RNA2 translation
RNA2 and Act1 levels (analyzed by qPCR)

control variables

WT (Saccharomyces cerevisiae) yeast strain
ΔYvc1 yeast strain transformed with an empty plasmid

controls

Positive control: WT (Saccharomyces cerevisiae) yeast strain
Negative control: ΔYvc1 yeast strain transformed with an empty plasmid

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