genophoria

Genomics euphoria: ramblings of a scientist on genetics, genomics and the meaning of life

Monthly Archives: July 2014

Letters from the trenches of war on cancer (Part I)

As I get older, cancer surpasses a scientific curiosity and morphs itself into a harsher reality. As our parents start to get worried about every mole and lump, we also accompany them through the ensuing emotional roller coaster. Working close to a hospital is not helping either… while the tumor samples you see every day are assigned random numbers, it is quite impossible not to see the human suffering behind every biopsy. While I still firmly and deeply believe in the fact that ultimately it is the basic research that can revolutionize health and medicine, I can also sense the urgency of now and the need to act on that front. It is this dichotomy that has shaped my research for the past few years, the fruits of which are finding their way into the annals of science.

It is not news to anyone that I study the biology and regulation of RNA (see the two previous posts on this very blog: here and here). I have specifically focused on developing computational and experimental frameworks that help reveal the identity of post-transcriptional regulatory programs and their underlying molecular mechanisms. Towards the end of my tenure as a graduate student, building upon the work by talented postdocs in the Tavaozie lab at Princeton University (namely Olivier  and Noam who published their work back in 2008) and with the help of my genius friend Hamed, we developed, benchmarked and validated a computational method named TEISER that extends motif-finding algorithms into the world of RNA by taking into account the local secondary structure of RNA molecules as well as their sequence.

When I started out as a postdoc, my goal was to study post-transcriptional regulation using cancer metastasis as a model. In addition to its clinical impact, studying metastasis also has the added benefit of access to a large compendium of high-quality datasets as well as rigorous in vivo and in vitro models for downstream validation of interesting findings.

When it comes to tumorigenesis in general, there is a large body of work focusing on the role of transcriptional regulation, specifically  transcription factors as suppressors and promoters of oncogenesis. However, other aspects of RNA life-cycle are substantially understudied. The success of our lab and many others in revealing novel and uncharacterized regulatory networks based on the action of various miRNA in driving or suppressing metastasis highlights the possibility that heretofore uncharacterized post-transcriptional regulatory programs may play instrumental roles in tumorigenesis.

Given the success of miRNA regulation and my previous work on RNA stability, performing differential transcript stability measurements between highly metastatic cells relative to their poorly metastatic parental populations seemed like a logical step. Using thiouridin pulse-chase labeling and capture followed by high-throughput RNA-seq, we estimated decay rates for every detectable transcript (~13000 transcripts total). It was around this dataset that we built an ambitious study, pushing ourselves to dig deeper at every step. We generated, analyzed, and interpreted heaps of data of various kinds: in silico, in vitro, and in vivo. The results of this study was the discovery of a novel post-transcriptional regulatory program that promotes breast cancer metastasis. Our results were recently published in Nature, however, I also gained insights that could not be included in a 4-page paper. As such, in the upcoming posts, I’ll try and expand on various aspects of this study that I found fascinating. Stay tuned…

RNA Structurome

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.

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.

Sources:

  1. Rouskin et al., 2014.  Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705
  2. Ding et al, 2014. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700.