Keystone always puts on some of the premier non-coding RNA meetings every year, and this was no exception. Our friend Athanasios Zovoilis from Harvard Med School made to trip out to Santa Fe and helped us cover the event. Check out his conference report:
The meeting focused on novel data on mechanisms governing long noncoding RNAs, which was also reflected in the opening Keynote Session that included a speech on long non coding RNAs from Dr. Phillip Sharp. Xist, as one of the few non coding RNAs that have been so extensively characterized in terms of function dominated many of the speaker sessions but the poster sessions included a substantial number of novel non coding RNAs, especially those originating from divergent transcription in promoter and enhancer regions.
This meeting included also a session on possible therapeutic applications of noncoding RNAs both as aptamers as well as potential chromatin modifiers. Interestingly, almost 1/4 of the sessions were devoted to bacterial RNAs and their catalytic properties and throughout the sessions there was an evident attempt to understand how findings in prokaryotes can reflect to higher organisms. In particular, many speakers noted the need to move from just describing the correlation of noncoding RNAs with specific cellular functions towards a more mechanistic insight into the RNA structures underlying these effects.
To this end, the poster sessions included many RNA structural assays, while a significant number of posters included extended RNA protein interaction assays of their novel RNAs with proteins that are chromatin modifiers. Finally, although the meetings scope did not associate long non coding RNAs with disease, in many instances (especially posters) the meeting extended also beyond mechanism, biochemistry and genome wide RNA and ChIP sequencing to include a wide disease spectrum that may be connected with these novel RNAs.
Synthesis and Functions of Noncoding RNAs
Phillip Sharp, MIT
During his keynote lecture, Dr Sharp presented briefly the current state of the art in the field of long noncoding RNAs, denoting that what had been only a small number of known non coding RNAs some years ago, has now extended to thousands of novel RNAs in this category. These findings have shed some light into the wide range of functions these RNAs may have but at the same time they had made it even more difficult at the moment to classify them into more distinct classes as in case of small RNAs. This is, in his opinion, one of the biggest challenges in the field together with understanding the biology related to each of these subgroups and any medical therapeutic applications.
Dr Sharp also drew our attention to two points that he believes to be the hottest topics at the moment in the field: The first is understanding the biogenesis and function of the recently identified circular noncoding RNAs. The second is revealing the impact of the so called super-enhancers that have been described recently. The latter according to Dr Sharp must be also seen under the light of the findings regarding pervasive transcription in the cell, and to this end he mentioned particularly the potential that GRO-sequencing offers towards this direction.
lncRNAs Target Chromatin Modifying Enzymes to Regulatory Gene Sequences
Ingrid Grummt, German Cancer Research Center
Dr Grummt’s talk discussed a rather new “unconventional” form of regulation by noncoding RNAs through the formation of triple DNA:RNA helix. Her talk focused on findings in the rRNA gene loci and particularly on the epigenetic state of these genes in response to external stimuli.
Starting with the question how are chromatin modifying enzymes guided to this region, she presented evidence that an RNA antisense to this locus may be a key player in this process. She showed that changes in the expression of this RNA correlate with response to serum deprivation and increased occupancy of rRNA genes by suppressive chromatin modifying enzymes. Interestingly, the recruitment of these enzymes to this locus seems to be realized by the formation of the above mentioned triple helix, which subsequently recruits DNMT3B.
The talk concluded with a series of experiments such as EMSAs regarding the formation of this triple helix and the exact element in the antisense RNA that is responsible for this interaction.
X-chromosome Inactivation and Epigenetic Regulation via ncRNA
Edith Heard, CNRS and INSERM
Dr Heard, who was one of the meeting’s organizers, presented her recent findings on the role of Jarid2 in X chromosome inactivation (XCI). Initially, Dr Heard referred to previous works of her lab and others on the way X inactivation differs from gene silencing occurring in the inactivated X chromosome.
This paradox could be explained according to Dr Heard if we take into account that PRC2 may participate in early maintenance of repression in X chromosome but not in initiation of its inactivation. Using Jarid2-GFP in female embryonic stems cells she went one step further and asked when is Jarid2 (which in many instances is part of the PRC2 complex) actually recruited during XCI. Interestingly, she showed that Jarid2 is transiently recruited in a chromatin localization pattern similar to that of Xist and Ezh2.
Dr Heard presented data showing that repeat A of Xist may not be critical for this recruitment in contrast to other repeats in Xist region. She then asked whether Jarid2 recruitment to inactive X chromosome is dependent on PRC2 and showed that knocking it down affects PRC2 recruitment through specific domains in Jarid2 protein. Finally, Dr Heard switched to in vivo experiments, particularly during pre-implantation and at the end of her talk proposed a model through which Jarid2 is recruited earlier than PRC2 to XIC.
ncRNAs in Bacteria
Ronald Breaker, Gisela Storz, Karen Wassarman, and Anna Marie Pyle
These talks included in this session as well as Dr Pyle’s talk in the Catalytic RNAs session were highly related and complementary and thus they are described here together. These talks made it clear that bacterial ncRNAs are a broad field unto themselves. By providing a number of examples of ncRNAs in prokaryotes these talks denoted that the divergence of functions of ncRNAs in bacteria can teach us a lot regarding ncRNAs in higher organisms both in setting the right questions as well as in selecting the right biochemical tools.
In brief, Dr Breaker (who was one of the meetings organizers) showed how large noncoding RNAs in bacteria can include structural domains with a catalytic activity. The presentation of an approach that led to a structured noncoding RNA discovery in bacteria dominated the first part of his talk, while the second part included particular examples of ncRNAs that were identified.
Dr Storz focused on the protein binding properties of bacterial ncRNAs, using RNAs bound to Hfq as an example. Dr Wassarman also presented another bacterial ncRNA, 6S RNA, that is bound by proteins (RNA polymerase) and affects their function. One from the most intriguing findings she presented was the use of this RNA as template for the synthesis of RNA.
Finally, Dr Pyle devoted the largest part of her talk to the significance of selecting the right conditions for RNA folding. Particularly, she emphasized that different ion concentrations and temperature protocols can be critical in acquiring an RNA structure more near to the real one in vivo, while certain interventions can be detrimental to this folding.
RNA Guided Genome Defense and Engineering by CRISPR Systems
Jennifer Doudna, University of California Berkeley
Dr Doudna’s work was of very high interest for the vast majority of the participants because although CRISPR was first identified in bacteria, its application in genetic engineering in mammalian cells makes it useful also when working with these model organisms. Dr Doudna presented in detail her approach to understanding how DNA interference works. presenting data on how this new viral spacer sequencing was acquired in the above system.
In the second part of her talk Dr. Doudna focused on Type II CRISPR systems and identified some key players in the recognition of DNA by Cas9/RNA complexes. Like other speakers in this conference, especially in the bacteria ncRNA field, she described the importance of understanding the RNA/protein structure underlying the CRISPR functions. Thus, her talk could have also been entitled “Towards a structural understanding of Cas9/RNA architecture”. According to Dr Doudna, the PAM element is central in this understanding, since it seems to construct one of the the basic mechanisms of DNA recognition by Cas9.
RNA Based Communication Inside and Outside Cells
Thomas Gingeras, CSHL
Dr Gingeras began with an update of the transcriptional status from ENCODE’s most recent data. According to Dr Gingeras it is of utmost importance to revisit the definition of “gene”. Based on his concept, the RNA transcript should be considered the most appropriate primary functional unit of the genome, while a gene is rather a higher order organizational concept which represents the collection of all transcripts and regulatory elements that correlate with phenotypic differences.
In the second part of his talk, Dr Gingeras described how the RNA content of extracellular vesicles may be critical to understand how cells may communicate with each other. Particularly, he presented evidence that different types of extracellular vesicles share different RNA content both in terms of average RNA length as well as sequence. Examples that he mentioned included exosomes and microvesicles and the groups of small and longer ncRNAs enriched in each of them.
A characteristic of this meeting not found in many previous meetings in this field, was the explosion in the number of structural and biochemical assays testing long non coding RNAs in terms of protein binding. As noted above, instead of just presenting novel ncRNAs within a specific biological context many groups presented mechanistic studies addressing the role of these lncRNAs in chromatin modification. To this end, the title of the meeting “marching towards mechanism” reflected in the best way the current trends in the field of long ncRNAs, as these were evident through the presented posters.
Perhaps the most interesting aspect of the conference was the diversity of the systems and methods by which ncRNAs are studied. Ranging from prokaryotes to eukaryotes, and from genetic to structural and basic biochemistry approaches, the talks and the posters revealed that the field evolves quickly in a rather inter- and multidisciplinary way. To this end, the future of this field promises to be both challenging and exciting.
**EpiGenie would like to give a huge thanks to Athanasios Zovoilis, MD, PhD who is a member of the Lee lab at Harvard Medical School, for providing this conference report**