The weekly or monthly updates that appear in my e-mail account from various journals that I have subscribed to serve as a reminder that every single day we are expanding our knowledge and adding to the repertoire of scientific conquest. Sometimes reading these papers, however, is a chore… Not every paper is well-structured, not every project deserves the attention that it receives, and not every study stands the test of time. Every now and then however, I read papers that leave a profound mark on how I view biological systems. These studies are not necessarily large-scale or even complex but the mere act of reading them changes my way of thinking. The transformation may be nuanced or not even noticeable, but the effects will remain… for a while. If pressed, each scientist may come up with a unique collection of such publications–what we find exciting is ultimately a subjective matter–but I think we all, to some extent, can appreciate the underlying attraction.
The late January issue of Nature carried a few papers of this type for me. Rouskin et al. and Ding et al. reported the use of DMS (dimethyl sulfate)-based modification of exposed ribonucleotide bases coupled with high-throughput sequencing to provide a snap-shot of RNA structural preferences in vivo (in yeast, mammalian cells, and Arabidopsis). Despite the need to overcome certain technical hurdles, the methods themselves are logical extensions of the methods that were published previously for low throughput and in vitro RNA structure determination. What I found intriguing, however, was how Rouskin et al. turned their observations into an actionable hypothesis. Given the nature of the data they had gathered, this paper could have easily turned into a descriptive publication. But the authors took a step further and put forth a hypothesis that best explained the major trends in their data. I am confident it would have been easier for them not to do so… I am also confident that because of this hypothesis, they had a harder time convincing the reviewers than they would’ve otherwise. But they clearly didn’t shy away from going were the data had taken them and they should be applauded for doing so. They put this hypothesis front and center; early on in their paper they state:
“Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermo-stable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells.”
For me, it all comes down to the phrase: “the importance of cellular processes in regulating RNA structure.” We have read about numerous examples where the structure of RNA acts as cis acting factors in RNA biology, however, thinking of RNA structure itself as an intermediate target of regulatory programs on a whole-transcriptome level is very intriguing. I always suspected this much but reading this sentence just toggled a switch in my head–in a good way.
DMS signal in RPL33A mRNA shows a region that is unstructured in vivo but forms a stable structure in vitro (Rouskin et al, 2014).
Based on their own DMS-seq data, Ding et al similarly report:
“…mRNAs of cold and metal ion stress-response genes folded significantly differently in vivo from their unconstrained in silico predictions (Fig. 4c, d and Extended Data Fig. 8a, b). Interestingly, these stresses are known to affect RNA structure and thermostability.”
This statement, despite being more descriptive, tells a similar story. And I think this is a very important hypothesis. Understanding RNA structure as a dynamic phenomenon in the cell, and not just a byproduct of thermodynamics coded within the sequence, with far-reaching regulatory consequences opens up a new field of research studying transcriptome-wide consequences of factors that affect RNA structure and their functional consequences.
I should also mention that in the same issue, a study by Howard Chang, Eran Segal and colleagues reported:
“Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS [–RNA secondary structure] information is encoded within RNA sequence.”
On the surface this statement contradicts those reported by the Weissman lab. However, this latter study was using de-proteinized RNA and as Rouskin et al. have clearly stated: “analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells.” So the observation made by Wan et al. is a consequence of the in vitro nature of their study. If it turns out that the differences between in vivo and in vitro RNA secondary structures are pervasive, as Rouskin et al. suggest them to be so, we need to rethink how much stock we’re willing to put into the descriptive studies that have reported on RNA structure using in vitro methods.
- Rouskin et al., 2014. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705
- Ding et al, 2014. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700.