Question

You speculate that there is a 4^th version of the eukaryotic RNA polymerase complex that you’ve wisely named RNAP IV. You think that it is responsible for transcribing protein coding genes only in cells exposed to SARS-CoV-2. Describe a process/set of experiments that would allow you to unequivocally demonstrate that RNAP IV is solely responsible for the production of mRNA after SARS-CoV-2 exposure.

Answer 1

experimental setup

1 control- containing scramble siRNA

2. Experiment- containing Si RNA against RNA pol I, pol II, pol III

both the control and experimental setup are kept under exposure of SARS COV-2. After transfection and checking for knockdown efficiency of the siRNAs via western blot, a part of the cells are analyzed by RNA sequencing.

If we observe a set of RNA transcribed equally in both the cases we can conclude that the RNA was formed by the polymerase IV. The other RNAs transcribed by other polymerases will be seen in the control setup but not in the experimental setup.

Also in another experiment,

in this case both the control and experimental setup have siRNAs against Pol I, II, III, and the control set has no infection while the experimental setup has SARS COV-2 infection. again RNA sequencing is done. In this case, if we observe a particular set of RNA to be transcribed in the experimental setup much more, we can say that that RNA transcription is dependent on the RNA polymerase IV which is activated only in SARS COV-2 infected cells.

These two experiments together prove that the RNA pol IV dependent genes are transcribed only in the presence of SARS-COV2 infection.