microRNAs: A picture may emerge
I finally got around to reading in depth the Science paper1 on how serum-starvation may cause microRNAs to increase the expression of their cognate genes. I’ve got to say that, at the very least, the paper got me thinking a lot about the mechanisms of microRNAs, since if this paper is right about upregulating genes with miRNAs, then the picture that has been built over the past few years may change dramatically.
First, a little scene-setting.
Lately, it seems like everyone wants to tie their research to microRNAs (a.k.a. miRNAs); they’re hot, they’re interesting, and it’s up-for-grabs really on how they work. Over the past few years, a lot of really vague and contradictory data has started to emerge about how microRNAs actually inhibit the expression of genes. Along with that, of course, are a whole swathe of theories, ranging from the idea that they work at the level of mRNA stability or translation to ideas that miRNAs recruit proteases that chew up proteins as they get made from the RNA.
A new review paper in Nature Reviews Genetics2 is out arguing for the idea that microRNAs stop translation initiation; other observations about microRNAs are either secondary consequences of this mechanism, or are minor, supplementary mechanisms for repression. It’s a very nice review, and it builds a very convincing case for their idea that the putative cap-binding activity of Argonaute 2 is one of the primary mechanisms for translational repression.
It seems to me like competition for 5′ cap binding could occur right after mRNA export and the putative pioneer translation round,3 which is thought to occur before the nuclear cap binding complex (CBC) gets replaced by the cytoplasmic eIF4F complex. If Ago2 manages to get its claws on the 5′ cap then, it’s certainly possible that eIF4F would never bind the cap, repressing translation, circularization, and even stabilization of the poly(A) tail.
(One of the problems with this theory, of course, is that non-capped RNAs, such as those with internal ribosome binding sites, can still sometimes be repressed by miRNAs (it seems). I’m not really sure how that fits into the larger picture; it’s entirely possible that these mRNAs are repressed by secondary mechanisms from miRNAs.)
Now, back to the paper I just read.
The cap-competition theory of miRNAs might explain the result of that paper in Science. The cool thing is that there is a paper from 2005 in Nucleic Acids Research4 showing that the cap binding of mRNAs by Poly(A)-specific ribonuclease (PARN) is at least partially serum-starvation dependent. So clearly, serum-dependent post-transcriptional regulation by cap-binding proteins has been seen before.
Now, this new Science paper purports that when cells are serum starved, miRNAs direct Ago2 to upregulate mRNA translation rather than depress it, like with others. It would be really interesting if these results pan out, because then they might unlock a cool and interesting piece of the miRNA puzzle, and it would definitely have vast implications for developmental biology, wound-healing, and overall gene regulation in the body.
The paper has some interesting experimental results, but there are a few fishy things about the paper that make its conclusions a bit forced. For one thing, the authors don’t really show miRNA-mediated repression of translation unless they synchronize their cells in their cell cycle. You can see this if you compare the expression levels of CX and CX with its cognate miRNA in the figure below (figure 2A from the paper):
As you can see, in the +Serum (grey) bars, CX and CX+miRcxcr4 have the same translation efficiency; the authors don’t see repression of CX when they transfected in miRcxcr4 unless they synchronize their cells (+Snc). (CX is an artificial sequence that they constructed with an artificial target site. miRcxcr4 is the artificial miRNA that targets CX.)
The problem here, of course, is that many other labs have seen repression of translation by miRNAs even when they don’t synchronize their cells. Thus, either the tons of other papers seeing this effect are wrong, or this one paper has some strange experimental quirks that may make the system very different from those of other labs (I’m inclined to think the latter). The authors argue (in the supplementary material) that perhaps other labs’ results are due to toxic effects of transfection reagents, or due to differences in cell culture protocols. It’s strange that so many other labs have seen such down-regulation without synchronization. Can every single one of those effects be an artifact? It seems unlikely.
So in the end, I’d like to see some sort of independent confirmation of the results of this paper before I’m likely to accept that somehow, almost the entire scientific community had screwed up their miRNA protocols and missed this crucial and drastically different role for miRNAs in post-transcriptional regulation.
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1. Vasudevan, S., Tong, Y., Steitz, J.A. (2007). Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation. Science, 318(5858), 1931-1934. DOI: 10.1126/science.1149460
2. Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?. Nature Reviews Genetics, 2008(2), 102-114. DOI: 10.1038/nrg2290
3. Chiu et al. (2004) Genes Dev 18, 745-754.
4. Seal et al. (2005) Nuc Acids Res 33, 376-387.
