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World Expert on RNA Interference (RNAi) and Small RNAs Gives Plenary Address on Final Day of 2015 Annual Meeting of International Society for Extracellular Vesicles (ISEV) in Washington, DC

On Sunday, April 26, the final day of the 2015 Annual Meeting of the International Society for Extracellular Vesicles (ISEV) (http://www.isevmeeting.org/), with a special focus on exosomes, the 800+ attendees were privileged to hear a plenary address from one of the world’s foremost authorities on small RNAs and RNA interference (RNAi), Gary Ruvkun (photo), Ph.D. Dr. Ruvkun is a molecular biologist at Massachusetts General Hospital and Professor of Genetics at Harvard Medical School. Dr. Ruvkun discovered the mechanism by which lin-4, the first microRNA (miRNA) discovered by Dr. Victor Ambros, regulates the translation of target messenger RNAs via imperfect base-pairing to those targets, and also discovered the second miRNA, let-7, and demonstrated that it is conserved across animal phylogeny, including in humans. These miRNA discoveries revealed a new world of RNA regulation at an unprecedented small size scale, and the mechanism of that regulation. Dr. Ruvkun has also discovered many features of insulin-like signaling in the regulation of aging and metabolism. Given that the cargo of exosomes and other extracellular vesicles frequently includes varieties of small RNAs, particularly miRNAs, Dr. Ruvkun’s world-class expertise in the field of small RNAs was particularly relevant to this ISEV audience. The winner of numerous prestigious science awards throughout his career, Dr. Ruvkun most recently was named a recipient of the 2015 “Breakthrough Prize in Life Sciences” (shared with Dr. Victor Ambros) for “the discovery of a new world of genetic regulation by microRNAs, a class of tiny RNA molecules that inhibit translation or destabilize complementary mRNA targets.” Dr. Ruvkun was warmly introduced to the crowd by brief remarks from Dr. Ken Witwer, Dr. Andrew Hill, and Dr. Marca Wauban, and then the eminent scientist took the stage. The title of his address was “The Tiny RNA Pathways of C. elegans,” but his talk also ranged far beyond C. elegans to describe a vast area of work in RNAi and to describe some recent developments in the field.

Initially, he outlined the process of RNAi in C. elegans, which he described as the “idiot savant” of RNAi as the organism is actually superb at executing this process.

In general, this RNAi process can be described as follows. An enzyme called Dicer cleaves long double-stranded RNAs into short ds RNAs (small interfering RNAs or siRNAs) of approximately 20 bps in length. These ds siRNAs are unwound into two ss siRNAs, known as the passenger strand and the guide strand. The passenger strand is discarded and the guide strand is incorporated into the RNA-induced silencing complex (RISC).

The most well-studied outcome of this is the post-transcriptional gene silencing that occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute (Ago) protein, the catalytic component of the RISC complex.

It was recognized early on that RNAi has a widespread role in silencing parasitic nucleic acids, such as those of mobile elements and certain RNA viruses, and thus contributes to the maintenance of genome stability and to the prevention of viral spread. It has been argued that, in organisms where mobile elements have decayed over long evolutionary times and have become nonfunctional, the RNAi pathway no longer offers a selective advantage and can be lost without major consequences.

Interestingly, Dr. Ruvkun remarked that C. elegans has a two-stage amplification process for this RNAi activity, as do certain other organisms, but humans have only a one-stage process. He further noted that worms such as C. elegans have 27 Ago proteins, while humans have just 8.

Furthermore, some organisms are able to carry out RNAi even though they have no Ago proteins at all. And some organisms do not have an RNAi pathway at all. This latter group includes the protozoans Trypanosoma cruzi and Leismania major. Many or all of the RNAi pathway elements are missing in some fungi, including the much-studied model organism Saccharomyces cerevisiae.

In his address, Dr. Ruvkun focused on work to investigate how RNAi is carried out in the absence of Ago protein. This involved comparing the C. elegans genome against the genomes of 86 animal, fungal, plant, and protist genomes to look for similar patterns that would suggest involvement in the same pathway, in this case the RNAi pathway, with the idea that Ago might be replaced by a different protein in different organisms in the flow of evolution.

The result of this work was that the top homologues found in this comparison were homologues of a gene called RDE-1 in C. elegans. This RDE-1 gene codes for the primary Ago protein in C. elegans. The identified homologues, Dr. Rukun suggested, are candidates for involvement in the RNAi process in organisms in which Ago has been lost.

He then moved on to a discussion of splicing factors, which are key to the process of removing introns from transcribed RNA and then rejoining the exon regions to form the mature RNA transcript. This splicing process takes place in particles called spliceosomes.

The exons can be spliced together in different ways, providing the cell with the capacity for producing multiple different proteins from the same gene. Dr. Ruvkun noted that humans have approximately 100,000 introns in their genomes, while S. cerevisiae, for instance, has just 100 introns in its genome.

He also suggested that this splicing mechanism is a “shrewd way to survey for infection,” because if foreign DNA is integrated into the genome, this foreign material will ultimately hinder the splicing process and cause the stalling of spliceosomes leading to a failure to produce the protein coding for by the infected gene. He further described this as a “serious anti-viral mechanism.”

He also noted the presence of two RNA-dependent RNA polymerase (RdRP) pathways that compensate for the absence of piRNAs in most nematodes. It has been suggested elsewhere that piRNAs in animals may have replaced an ancient eukaryotic RdRP pathway to control transposable elements.

Dr. Ruvkun also noted that the enzyme enolase is found in the RNA degradosome of many bacteria and that this suggests a possible connection between the glycolytic pathway and RNAi.

He then moved on to a discussion of “mutator genes.” These are genes that code for proteins that can still recognize and inactivate invading nucleic acids and are also indicators of past invasions. These mutator genes play key roles in RNA surveillance. Dr. Ruvkun noted, in particular, the mutator gene mut-16, which is a prion-related gene that is required for genes that yield the most siRNAs.

Dr. Ruvkun described studies in which the mut-16 proteins, as well as other mutator proteins were shown to localize just adjacent to the cell nucleus, around its periphery. Furthermore, he showed that mut-16 localizes to the germline.
Then, as he concluded his presentation, Dr. Ruvkun again acknowledged how awesome C. elegans is at the process of RNAi, but said that, nevertheless, he is hoping to further improve on that process and is currently working hard on that pursuit in the laboratory.

Certainly, this wide-ranging and highly informative address on small RNAs will prove enormously useful to many of the exosome researchers in the audience as they continue their work on these tiny, but ubiquitous and powerful vesicles that seem to exert many of their yet-to-be-completely-defined functions via small RNAs that often constitute their cargo.

With that, the scientific sessions of the meeting came to an end and the audience was treated to a brief, but highly stimulating closing ceremony led by ISEV president Jan Lötvall.