what is the possible mechanism for a downregulation of RNA expression - (Dec/16/2013 )

hi everyone, I am working on the interation between a viral protein A and host protein B. I found that this viral A protein inhibits mRNA expression of host B protein by real time PCR. As always, I cloned the promoter of B (1.5kb upstream of TSS) to a Luc reporter and transfected it to a B-high-expressing cell line. however, I did not see any inhibition. Although it looks like this reporter might be OK because I saw a 10-fold increase compared to the Luc reporter without insert. I dont have a reliable positive control but I used chemical treatment which I have confirmed to downregulate B mRNA expression. This chemical also did not inhibit the promoter activity. Instead, both induced a bit increase.

 

I know it is still likely that my promoter reporter was not good. But what if it was, then what exactly downregulated the mRNA of B. Do you have any idea regarding the regulation of mRNA? Any comment would be appreciated. Thanks.

It could be causing the RNA to be degraded by miRNA or shRNA expression.  There could be a degradation of the RNA by other pathways.  It could be physically stopping transcription, or causing the RNA pol to be bumped off the promoter.

 

I cloned the promoter of B (1.5kb upstream of TSS) to a Luc reporter and transfected it to a B-high-expressing cell line

I presume you mean "high A expressing cell line"?

bob1 on Mon Dec 16 19:45:32 2013 said:

 

It could be causing the RNA to be degraded by miRNA or shRNA expression.  There could be a degradation of the RNA by other pathways.  It could be physically stopping transcription, or causing the RNA pol to be bumped off the promoter.

 

 

 

I cloned the promoter of B (1.5kb upstream of TSS) to a Luc reporter and transfected it to a B-high-expressing cell line

I presume you mean "high A expressing cell line"?

 

Thank you very much for your reply. I did mean high B expressing cells because I thought the promoter of B might be active in that cell line so that I can expect a reduction of my reporter activity when coexpressing A.

 

I have further questions that I really hope you can answer me.

 

1. You pointed out the possibility of RNA degradation. In this regard, what is the best way to study it (non-Northern-way)?

2. If the degradation is caused by stopped transcription or bump-off of RNA pol from the promoter, isn't there a decrease in the promoter activity?

 

Thank you very much Bob1.

gyma on Tue Dec 17 02:18:33 2013 said:

Thank you very much for your reply. I did mean high B expressing cells because I thought the promoter of B might be active in that cell line so that I can expect a reduction of my reporter activity when coexpressing A.

 

I have further questions that I really hope you can answer me.

 

1. You pointed out the possibility of RNA degradation. In this regard, what is the best way to study it (non-Northern-way)?

2. If the degradation is caused by stopped transcription or bump-off of RNA pol from the promoter, isn't there a decrease in the promoter activity?

 

Thank you very much Bob1.

1. You could input labeled RNA (stable isotope? radiolabel) and do some sort of pulse-chase, but this would require a method of specifically detecting the RNA, for which northern would be good.  You could also use some inhibitor of the normal RNA degradation pathways (if there is such a thing).

 

2. Stopped transcription could potentially occur anywhere in the transcript before the region you used for your PCR primers.  Bumping off the promoter would lead to a decrease in promoter activity.

 

I see what you mean by the high B expressing cells, just me confusing myself.  I would go for a line that has a relatively low expression of B if possible - otherwise you have to overcome the effect of having lots and lots of B promoting stuff in there before you will see a change in the Luc activity.

Thank you very much for these valuable advice.