Comparative RNA Structure Analysis of Nascent and Mature Transcripts in Saccharomyces cerevisiae

We studied cotranscriptional RNA processing, and that includes how nascent RNA folds as it emerges from the RNA polymerase that synthesizes it. Before this method, we were not able to monitor how RNA folds during synthesis, and this was holding us back in our research. So this new method overcomes those gaps.

CoSTseq was developed to investigate nascent RNA folding in yeast. It is particularly effective for analyzing highly abundant RNAs. To begin, inoculate Saccharomyces cerevisiae strain BY4741 in 50 milliliters of YPAD media and incubate at 30 degrees Celsius with shaking at 200 revolutions per minute until the culture reaches mid-log phase.

Collect approximately three milliliters of yeast culture corresponding to 1.8 optical density units and centrifuge at 2, 500 x g for three minutes at four degrees Celsius using a pre-cooled centrifuge. Discard the supernatant into a waste container. Resuspend the pellet in 10 milliliters of cold PBS and centrifuge again at 2, 500 x g for three minutes at four degrees Celsius.

Remove the supernatant and keep the yeast pellet on ice. Carefully resuspend the yeast pellet in 10 milliliters of cold 0.5%sarkosyl without creating bubbles. Incubate the resuspended yeast on ice for 20 minutes to allow permeation, then pellet the cells at 400 x g for five minutes at four degrees Celsius and resuspend the permeable yeast cells in 100 microliters of nuclease-free water using a P-1000 pipette.

Prepare a working solution of 2.5X transcription buffer with freshly added five millimolar dithiothreitol and preheat 2.5X structure probing buffer to 30 degrees Celsius. In a clean two milliliter tube, add all the required reaction components and mix thoroughly. Next, add 100 microliters of the prepared yeast cells to the reaction tube and incubate it in the thermo mixer set to 30 degrees Celsius, with shaking at 500 revolutions per minute for two minutes.

Then quickly add 200 microliters of the pre-warmed 2.5X structure probing buffer, and 25 microliters of dimethyl sulfate reagent simultaneously. Vortex the tube gently in two pulses and place it back in the incubator set to 30 degrees Celsius and 500 revolutions per minute. Continue to incubate on the thermo mixer for four minutes, spacing 30 seconds between samples.

Prepare the stop and wash buffers ahead of time and place them on ice until use. Stop the dimethyl sulfate methylation by adding one milliliter of stop buffer to the reaction tube. Now centrifuge the dimethyl sulfate labeled samples at 3, 500 x g for five minutes in a pre-cool centrifuge.

After the spin, discard the supernatant into an appropriate waste container. Add one milliliter of chilled wash buffer to the pellet and resuspend. Repeat the centrifugation at 3, 500 x g for five minutes.

Proceed immediately to RNA extraction and do not freeze the yeast pellet. Resuspend the dimethyl sulfate labeled yeast pellet in 600 microliters of RNA lysis buffer, and then transfer the suspension into a tube containing 40 microliters of 20%sodium dodecyl sulfate. Incubate the sample at 65 degrees Celsius for 30 seconds, with shaking at 950 revolutions per minute.

Next, perform phenol-chloroform extraction of RNA following the standard procedure. Vortex the streptavidin magnetic beads and transfer 44 microliters of the beads to a new tube for each sample of 80 micrograms total RNA. Place the magnetic beads on a magnetic rack until the beads settle.

After removing the storage buffer, resuspend the beads in one milliliter of pre-wash buffer A.Now incubate the suspension for two minutes at room temperature, then magnetize and remove the supernatant. After washing, resuspend the beads in 88 microliters of 2X binding buffer. Transfer 80 microliters of the suspension to a sterile 1.5 milliliter tube containing 80 micrograms of biotinylated RNA sample in 80 microliters.

Rotate the bead sample mixture on a rotator for 20 minutes at room temperature. Then place the tube on the magnetic rack to collect the flow through containing mature RNA and save it for precipitation to perform poly-A selection of mature RNAs using DMS-MaPseq workflow. Next, rinse the bead-nascent RNA complex twice with 500 microliters of high salt buffer.

Then rinse once with 500 microliters of 1X binding buffer, followed by a final rinse with 500 microliters of low salt buffer. Now add 300 microliters of RNA reagent to the bead=nascent RNA complex, resuspend thoroughly and incubate on the thermo mixer for five minutes at 60 degrees Celsius. Next, add 60 microliters of chloroform, vortex and incubate for three minutes at room temperature.

Centrifuge the sample at 14, 000 x g for five minutes in the pre-cool centrifuge. Finally, transfer the upper aqueous phase approximately 180 microliters to a new collection tube. Discard the remaining organic phase, leaving behind the beads and residual aqueous solution.

After purifying the nascent RNA, perform template switching reverse transcription, ligate the 5'adapter and select libraries for sequencing. Visualization of the test PCR products on a 1%agarose gel revealed minimal primer dimer formation and a characteristic smear around 300 base pairs for co-transcriptional structure tracking sequencing or CoSTseq and DMS-MaPseq libraries. The electropherogram for CoSTseq sample one confirmed the library size distribution with a peak around 285 base pairs.

Read alignment of ASC1 mRNA revealed that the CoSTseq library included coverage across the intron, confirming that the RNA is nascent. Whereas the DMS-MaPseq library lacked intron coverage, indicating that the RNA is mature. The cot transcriptional folding matrix of 18S pre-rRNA revealed that the DMS reactivity showed abrupt changes as RNA polymerase progressed from position 790 to 811 along the rDNA locus.

DMS reactivity analysis of ADH1 mRNA showed similar profiles between nascent and mature forms, indicating regions of structural stability. In contrast, SSA2 mRNA exhibited regions of different DMS reactivity between nascent and mature forms, suggesting structural changes during maturation. With CoSTseq, researchers can determine in vivo nascent RNA secondary structures and the position of RNA polymerase along RNA transcripts.

CoSTseq has time-sensitive steps, uses toxic chemicals. It is better to process no more than two samples at a time. From the total RNA generated from CoSTseq, non-biotinylated RNAs can be used for concurrent mature structure probing using traditional DMS-MaPseq.