RNA Journal Club 9/3/09
Wenqian Hu, Thomas J. Sweet, Sangpen Chamnongpol, Kristian E. Baker & Jeff Coller
Nature 461 (7261): 225-229, 10 September 2009.
Nature AOP, 23 August 2009.
This week’s exacting summary and analysis by David Weinberg:
In their recent Nature article, Jeff Coller and colleagues demonstrate that mRNA in decay in the budding yeast Saccharomyces cerevisiae can occur while the mRNA is still engaged with actively translating ribosomes. Prior to this paper, the dogma in the field had been that ribosome dissociation was a necessary step before decapping. The evidence for this exact model was a bit lacking: it was clear that translation initiation and decapping are competing processes since both require access to the cap structure, but there didn’t seem to be any indication that decapping would have to interfere with elongating ribosomes. Indeed, co-translational mRNA decay had been previously hypothesized but no lab had demonstrated it. Using the tools of budding yeast and some clever molecular biology, Jeff Coller’s lab is able to do just that.
The basic outline of the article is a series of very similar experiments showing that decaying mRNAs (i.e., deadenylated, decapped, and/or partially degraded) are associated with translating ribosomes using different combinations of knock-out and wild-type strains, and artificial and endogenous mRNAs. The initial indication that the existing model for mRNA decay might be incorrect is that deadenylated mRNAs that accumulate in a decapping-defective strain remain on polyribosomes. The same is true of decapped mRNAs that accumulate in an XRN1 knock-out strain. The authors are careful to include enough control experiments to show that the polyribosome association suggested by sucrose gradients is actually due to bound ribosomes and not another macromolecular complex that might similarly alter the sedimentation properties of an mRNA.
Next, using both transcriptional turn-on and shut-off experiments the authors claim to show that decapping can occur when mRNAs are associated with translating ribosomes. This point – that the associated ribosomes are actively translating when decapping occurs – is important to distinguish from ribosome-reloading following mRNA decapping. The turn-on experiment is a bit bogus as their interpretation is based on the observation that at the first timepoint when a decapped mRNA accumulates, that decapped mRNA is on polyribosomes. Given that they had 2 timepoints (20 and 60min), this conclusion seems like a stretch and could have been left out of the paper without any detriment. However, the transcriptional shut-off experiment is convincing: adding cycloheximide at the same time that transcription is shut-off prevents the ribosome run-off on decapped mRNAs that is seen in the absence of cycloheximide. Thus, the ribosomes observed to be associated with decapped mRNAs were in the act of translation.
Up to this point, all experiments had been performed in mRNA decay mutants in order to observe relatively rare decay intermediates. Using an artificial mRNA in which rare codons are used to stall translating ribosomes, the authors demonstrate that their conclusions also hold in wild-type cells. Of course, such an artificial mRNA can potentially give results that aren’t true of endogenous mRNAs that are efficiently translated. The authors therefore conclude their paper with the ultimate demonstration of co-translational mRNA decay for two different (highly expressed) endogenous mRNAs in wild-type cells.
From their work, the authors conclude that a ribosome-free state is not required for mRNA decay and, in fact, mRNA decay can occur co-translationally. This ability to initiate decay of mRNAs that are still being translated seems to provide a more rapid means of mRNA decay: rather than waiting for translation to finish or ribosomes to be actively removed, Xrn1 can begin to degrade the mRNA from the 5′ end immediately after decapping. The paper also makes an evolutionary argument that such a decay mechanism would have the benefit that it would not interfere with residual translating ribosomes and, therefore, would prevent the production of truncated polypeptides.
While the science in this paper is (for the most part) convincing, its presentation is a bit frustrating. At the end of the paper, the experiments that the reader should really care about – those performed with endogenous mRNAs in wild-type cells – are FINALLY shown. But to get there the reader has to get through the artifact-prone experiments done in other settings. Of course, these artifact-prone experiments lend further support to the final model. However, I would have rather seen some space in the paper devoted to further characterization of the decay mechanism. For example:
– Do the same principles hold for all mRNAs? In particular, is this true of histone mRNAs that lack poly-A tails? Lower expressed mRNAs that are more difficult to detect?
– Is Xrn1 decay of ribosome-associated mRNAs distributive or processive? Are there cycles of degradation/dissociation as ribosomes finish elongating, or does Xrn1 remained engaged throughout?
– How does mRNA half-life relate to ribosome occupancy? Is slow translation of an mRNA associated with slow decay?
I hope that we can find some answers to these questions in future work from the Coller lab.