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I have seen a lot of research into molecular mechanisms of diseases/phenotypes use measures of RNA as a 'proxy' for the level of protein available in the cell. Is this actually valid?

My problem with the assumption that RNA levels correlate with that of the active product (i.e. the protein) is that a lot of post translational regulation occurs, including co-factor binding and phosphorylation, to name but 2. Does anyone know of any studies that have looked into the correlation between RNA levels and protein levels, and separately into the correlations between RNA levels and active protein?

It makes sense to me that RNA would correlate with protein certainly, but whether this relates to the proteins active function is what I wonder - i.e. there could be a pool that is replenished as and when the protein levels drop, but the proteins are only actually active for short periods in response to specific stimuli. So, does anyone know of any studies that have looked into the correlation between RNA levels and protein levels, and separately into the correlations between RNA levels and active protein?


Update (04.07.12)

I have not accepted any answers as yet because none address my question about levels of protein activation, but I concede to Daniel's excellent point that proteins are not all activated in the same way; some are constantly active, some require phosphorylation (multiple sites?), some binding partners... etc! So a study looking at 'global' activation is not yet possible. Yet I was hoping that someone may have read some specific examples.

I today found an unpublished review by Nancy Kendrick of 10 studies that have looked at the correlation between mRNA and protein abundance - still not relating to activation. However she finishes the paper as follows;

The conclusion from the ten examples listed above seems inescapable: mRNA levels cannot be used as surrogates for corresponding protein levels without verification.

If this is her conclusion about protein levels, then any correlation between protein activation and mRNA abundance seems unlikely (as a rule. Some protein levels do correlate with the RNA - see the paper).

I am still interested in any answers that give any information about specific examples of protein activation and mRNA levels - it seems highly unlikely there are no such studies, but I have been as yet unable to find any!

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Good point: also you can imagine that if some mutation results in (for example) protein X being ubiquitylated you'd be likely to see an increase in the RNA that codes for X while the level of X drops. In this case the RNA would make a pretty lousy proxy. –  Shep May 10 '12 at 13:24
    
To be frank, if you can't find a correlation between mRNA and protein abundance, why would you bother to ran the next experiment to find correlations between mRNA and protein activation? –  bobthejoe Jul 5 '12 at 7:35
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@bobthejoe it does seem like it would be a fruitless experiment - doesn't mean no one has looked though (in fact I am surprised there isn't more literature on it). If no one else has found any then I will either accept one of the answers already given or post my own combining the evidence against any correlation. –  Luke Jul 5 '12 at 8:23
    
@LukeTheDuke, I'm cautiously excited about your hypothesis. –  bobthejoe Jul 5 '12 at 22:07
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2 Answers 2

It has been well established that mRNA abundance serves as a poor proxy for protein abundance in most cases. This paper on yeast and this paper on cancer both establish this, although using older techniques (SAGE and microarrays, respectively), while this more recent review discusses the topic in light of more recent technologies (e.g. RNA-seq).

Perhaps the difficulty with exploring the correlation between mRNA levels and levels of active, mature proteins is that we still know so little about so many proteins and and what makes them active and mature versus inactive, premature, etc. Not all proteins need post-translational modifications to be active, but some do. Currently, this is investigated on a very detailed protein-by-protein basis (as far as I know), so gathering enough data for a large-scale study could take a long time. On the other hand, there are a variety of (increasingly affordable) high-throughput methods for measuring the abundance of thousands of RNA species simultaneously. I think people are using mRNA levels to estimate expression not because it's the most biologically cogent course of action, but rather because it is much easier, more affordable, and more high-throughput (which is all the rage these days).

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It's interesting that the RNA doesn't even correlate well with protein abundance it that example, let alone activation state. I agree that the reason RNA is measured is for the comparative ease with which its abundance is quantified, however my issue is with the conclusions drawn in many papers based on the assumptions that RNA levels are at least indicative of protein abundance/activation! Thanks for the links and your reply –  Luke May 10 '12 at 13:53
    
@Daniel: +1 for ending it at "..high throughput (which is all the rage these days)"! –  gkadam May 10 '12 at 16:19
    
Sometimes, overproduction of mRNA is a response to low concentrations of the expressed protein. Great answer. –  bobthejoe May 10 '12 at 17:32
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I think that overproduction of mRNA in response to low protein is potentially what studies are looking for, as it implies the protein is in demand at that time (compared to a protein that does not need replenishing). This is of course confounded by that fact that proteins will have different lifespans! Thanks for your answer, I am hoping for more answers that might address the link between mRNA and protein activation, if there is any at all. If there isn't, then I fear that many studies may be reporting false claims! –  Luke May 11 '12 at 12:51
    
I think you'll find very few papers that demonstrate changes in mRNA levels by microarray, claim that an increase in the gene product's activity is responsible for the biological effect, and then stop. mRNA data is complicated, not useless. Almost anywhere that mRNA upregulation is reported, it will be followed by a measurement of the protein levels and other follow-on experiments to confirm the protein's involvement. –  Transcriptase May 11 '12 at 15:11
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This study in E.coli is useful in setting out some additional issues at play here, in a system where post-translational modifications are basically a non-issue. The authors see clearly a correlation between mRNA and Protein levels (Fig. 3C) and theorize that differences translation rate and protein turnover are responsible for differences in protein level for two genes with similar mRNA level.

They investigate the issue on a single-cell level and find no correlation between a single cell's mRNA level and protein level for a given gene (Fig. 4). This is attributed to the time-scale separation between mRNA turnover (usually a few minutes) and protein turnover (in many cases degradation is so slow, protein turnover is driven by dilution/cell division). One way to understand this is there are several "generations" of mRNA contributing to a the population of protein at a given moment, which is not inconsistent with the central dogma. However this result raises serious problems for using mRNA as a proxy for protein in comparing single-cells.

However generally, in populations of bacteria, using mRNA as a proxy for protein works pretty well, especially when comparing levels of a gene under perturbations. Unless we believe translation or protein degradation is affected mRNA level should be a good proxy, though there can be issues with using this quantitatively because regulation is often not linear (i.e. double mRNA doesn't produce double protein). See, for example, sRNA regulation.

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Thanks for the answer, and link to the study. It makes sense that mRNA and protein levels correlate to some degree, for the reasons you mention there will never be complete concordance. I am interested to know of any studies that have looked into whether mRNA levels are at least indicative of protein activation - I am particularly interested with regard to the relevance to humans. Many thanks. –  Luke Jun 8 '12 at 14:43
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