Modulation of gene expression through promoter induced nuclear RNA degradation
Description
The data describes the analysis of Axl2 mRNA localization during the cell cycle using FISH and lives cell assay as well as Northern blot analysis of the mRNA abundance and cleavage by yeast RNase III Rnt1p. Three sets of data are included: 1) FISH images, 2) Live cell analysis images and 3) Images of Northern blots
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Steps to reproduce
The methods are described in the accompanying papers FISH The yeast FISH protocol was performed essential as previously described before (Trcek et al., 2018). 50 mL cultures were grown to OD600 of 0.4 to 0.6 and fixed for 45 min at room temperature by adding 32% electron microscopy grade formaldehyde to a final concentration of 4%. Fixed cells were washed with buffer B (1.2 M sorbitol, 100 mM KHPO4 pH 7.5), digested for 10 to 20 minutes in buffer B with 20 mM vanadyl ribonucleoside complex, 20 mM beta-mercaptoethanol and 125 to 375 U of lyticase adjusted for each strain to achieve >90% cell digestion in that time frame. Digested cells were washed with buffer B and stored at -20°C in 70% EtOH. 1.5E7 cells were rehydrated in 2X SSC and prehybridized in 2X SSC 10% formamide for 1 hour at 37°C. Hybridizations were carried in tubes over night at 37°C in a final volume of 100 μl containing 2X SSC 10% formamide 10% dextran sulfate and 10 ng of each sets of probes. Probes sets consisted either of 12x 20 nucleotides long DNA oligos complementary to the 5’ sequence of Axl2 mRNA labeled with DyLight 650, 12x 20 nucleotides long DNA oligos complementary to the 5’ sequence of Axl2 mRNA labeled with DyLight 550 or 4x 20 nucleotides long DNA oligos complementary to the internal spacer 2 sequence of the pre-ribosomal RNA labeled with Alexa 488 used as an hybridization control. Probes sequences were chosen using the Stellaris Probe Designer web tool and synthesized by Biosearch technologies. After hybridization, cells were washed once with 2X SSC 10% formamide, once with 2X SSC 0.1% triton, and once with PBS. 2E5 stained cells were deposited on poly-lysine coated coverslips, allowed to settle for 30 minutes and washed with EtOH prior to mounting on slide with ProLong Gold with DAPI. Slides were sealed after an overnight curing at room temperature. Visualization was performed on a Zeiss microscope with an oil immersion alpha Plan-Apochromat 100x/1.46 objective. A Zeiss mRM camera was used and z-stacks of 20 images spaced by 200 nm were collected using the Zen 2012 software. DAPI was illuminated for 200 ms with a 365 nm LED and viewed through filter cube 62HE, DyLight 550 was illuminated for 2 seconds with HXP lamp and viewed through filter cube 43HE. DyLight 650 was illuminated for 4 seconds with a 625 nm LED and viewed through filter cube 77HE. For display purpose, the image stacks were deconvolved using Zen 2012 constrained iterative algorithm and the maximum projection is presented in the pictures. Live cell imaging Yeast cultures expressing MS2 repeats in the 5’ UTR of Axl2 mRNA and a plasmid containing MCP-GFP and NLS-td-tomato were grown to an OD600 of 0.4 to 0.6, concentrated by centrifugation to an OD600 of 2 and spotted on 1.2% agarose pads made with growth media. The coverslips were sealed with petroleum. Slides were visualized in a 26°C incubation chamber on a Zeiss spinning disk microscope with an oil immersion alpha Plan-Apochromat 100x/1.46 objec