Protein expression opposite of mRNA expression. Ideas? - (Feb/01/2013 )

I have interesting results and I'm not sure where to go from here.

I have a cell line stably transfected with the wild type gene, and two mutated and constitutively active versions of the gene. The mRNA and protein expression of the gene is very high compared to the naive cells. So all is well with the cell lines.

I identified a gene whose mRNA expression is greatly reduced as a result of the two mutant forms of the gene compared to the transfected wild type and naive cells. It was reduced by at least hundred(s) of folds. It is a significant reduction. This was confirmed via microarray, RT-qPCR and Northern blots. I'm pretty confident in these results.

When I examined the protein expression of this identified gene, the mutated transfected cell lines expressed this gene at a much higher rate than the wild type gene. The wild type transfected cell line had reduced expression of the protein compared to the naive, and the mutated transfection doubled the protein expression. I've repeated these Westerns a few times. Again, I'm pretty confident in this part as well.

So in summary, mRNA indicates the gene is suppressed in mutant cell lines. Protein indicated the gene is increased in the mutant cell lines.

One problem here is that the regulation and signalling upstream of this gene isn't well known at all. I don't exactly have specific targets to go after.

I haven't had experience with this... anybody have any ideas/suggestions/places to look at? Any feed back would be most welcome.

Thanks!

The main problem is that you are working off transfected cell lines - unless you can be sure that the promoters are mammalian and that you only have single copy insertions for each gene/mutant then you can't really compare results for RNA or protein expression.

bob1 on Fri Feb 1 22:41:58 2013 said:

The promoters are mammalian, and I can't control for the number or location of the viral insertions. They are random, can't be helped.

But how does that effect a downstream gene/protein having completely inverse protein and mRNA?

I guess a more generalized question would be to remove the transfections from the equation. What could cause the protein and mRNA expression to be completely inverse of one another in a cell?

Probably miRNAs... anything else?

Could be that the protein being expressed off the mutant mRNA actually binds better to the antibody than the wild-type protein. How are you detecting the mutant and the wild-type?

To clarify, the protein/mRNA inverse relationship is NOT in the tranfected gene. The transfections worked well by all indications. It shows up well in the DNA and the transfected gene is HIGHLY over expressed at the mRNA level, and over expressed at the protein level.

The inverse mRNA/protein is observed in a gene that appears to be downstream of my transfected gene.

My working theory is that some miRNA is blocking translation but not degrading the mRNA.

Ah, OK that makes more sense. It could be that the mutant form of the protein is more stable (e.g. no/fewer lysines for ubiquitination), or that there is a structural change that causes the protein to remain in the cell.

You may be interested in a recent article by Carrieri et al in Nature http://www.nature.com/nature/journal/v491/n7424/full/nature11508.html
They showed that a lncRNA could act as a chaperone increasing translation efficiency. In their experiments, the mRNA levels were unaffacted but the protein levels increased - similar to what you are seeing. I'm not sure if that is what is going on with your downstream gene, but it may offer an alternative explanation. If you want to try using their technology yourself, its available from Cell Guidance Systems,