A summary of 2025 projects:

Small RNA biology

Small RNA pathways regulate eukaryotic defense against invading genetic elements such as viruses.  RNA interference in C. elegans, plants, and fungi uses Argonaute and RNA dependent RNA polymerase proteins for silencing of transposons and other integrated viruses as well as new invading viruses.  The new genes we have discovered that strongly enhance RNAi reveal mechanisms by which siRNA production is naturally broadened to defend against novel viruses.  Our saturation genetic analysis of antiviral pathways has revealed new possibilities for how the genomic record of past viral infections can anticipate the constant variation in new viruses that are encountered, using DNA and RNA-based recombination between viral genomes archived in the C. elegans genome and RNA editing of those genomes.

Somatic misexpression of germline meiotic recombination and RNA interference genes in dREAM complex mutants (Wang Ruvkun 2005; Wu Ruvkun 2012; Fischer Ruvkun 2013).  Our comprehensive screens for RNAi defective and for enhanced RNAi mutants and gene inactivations have revealed an intersection between regulation of natural gene endoreduplication and RNAi in C. elegans.  A large fraction of the gene inactivations that we discovered to enhance RNAi were also isolated as synMuv B genes in genetic screens in the Horvitz and Han labs for increased EGF signaling in vulval patterning.  We discovered that most of the synMuv B mutants strongly enhance RNAi and cause misexpression of germline-specific genes in the intestine, including many genes implicated in RNAi such as pgl-1 and pgl-3 in P-granules.  These dozen synMuv B genes encode homologues of the mammalian dREAM complex found in nearly all animals and plants, including tumor suppressor Rb. The Drosophila dREAM complex binds specifically at replication origins that flank the Drosophila chorion genes to control their local (about 50kb) endoreduplication.  Gene expression analysis  of C. elegans dREAM mutants reveals that many normally germline-specific genes are markedly upregulated in the soma (Kirienko Fay, 2006; Petrella Strome 2011; Wu Ruvkun 2012).  There is a common theme to all of the processes affected by the dREAM complex: the C. elegans intestine and hypodermis (Hedgecock White 1985), like the chorion of Drosophila (Royzman Orr-Weaver 1999, Bosco Orr Weaver 2001) undergo multiple programmed full genome endoreduplications that are probably independent of the dREAM complex but then a highly localized dREAM complex-mediated controlled amplification to dramatically increase gene dosage of only those 50 kb scale genomic regions.  Our full genome RNAi screens for gene inactivations that disable RNAi (Kim, Ruvkun, 2005; Wang Ruvkun 2005) revealed additional candidate regions for localized endoreduplication to increase RNA interference:  we found that 6 of 36 viable RNAi-defective gene inactivations tested can suppress the enhanced RNAi and the misexpression of P granules in dREAM complex mutants.  Amazingly, those same RNAi-defective suppressors of enhanced RNAi in synMuv B mutants also suppressed the Multivulva phenotype (too much EGF signalling) from the hypodermis, a tissue that also endoreduplicates in C. elegans (Hedgecock White 1985).  In support of a DNA amplification model, the human homologues of many of the suppressor of synMuv B genes are often translocation breakpoints or amplified regions in tumors.

In the 32C polyploid intestinal cell that will never divide again, there is magnificent ammunition for natural generation of genetic diversity by unequal crossing over in repetitive elements.  Thus we are now testing whether the C. elegans dREAM complex mutants mediate localized amplification of client genomic regions such as the holocentromeric elements that we hypothesize mediate antiviral defense.  In the synMuv B mutants, chromosome synapsis and recombination genes are strongly upregulated, favoring a model that recombination may be activated.  The unequal crossing over of tandemly arrayed repeated holocentromeric elements is an excellent candidate to generate siRNA diversity.  We are searching for these recombinant siRNAs in the synMuv B mutants.

 Phylogenetic profiling to discover genes that act with Argonaute and RNA dependent RNA polymerase genes to mediate antivirus programs. Tabach Y,..G Ruvkun 2013.  Nature 493:694-8. Sadreyev R, Ruvkun G, Tabach Y. 2015.  Nucleic Acids Res.  43:154-9. Argonaute/PIWI proteins process and present small RNAs to their targets.  Argonaute proteins are widely distributed across animals, fungi, plants, and protists; the basal eukaryote had Argonautes and was probably competent for RNAi.  But Argonautes and the competence for RNAi have been lost in about 30% of Ascomycota and some protists.  To identify other genes that were lost coincidently with small RNA pathway genes, we determined the phylogenetic profiles of all 20,000 C. elegans proteins in 85 animal, fungal, plant and protist genomes.  Some genes that have similar phylogenetic profiles to Argonautes but bear no homology were: multiple RNA splicing factors and a number of coenzyme A metabolic genes.  We found that about half of the small RNA candidate genes predicted by phylogenetic profiling are required for RNAi silencing in genetic tests for RNAi competence.   To identify other genes that function in the same pathways as small RNAs and therefore are lost coincidently with small RNA pathway genes, we determined the phylogenetic profiles of all 20,000 C. elegans proteins in 85 animal, fungal, plant and protist genomes. 

            The RdRp genes are central to RNAi in C. elegans, fission yeast, and plants, mediating the secondary amplification of siRNAs generated by Dicer and Argonaute proteins in the first stage of RNAi. The secondary amplification of primary siRNAs by RdRps, is a powerful amplifier of RNA interference capacity;  It is not a coincidence that RNAi was discovered somewhat synchronously in worms, plants, and fungi, all of which encode RdRps that mediate secondary siRNA amplification, unlike the less RNAi proficient mammals for example. But RdRps are also key RNA replicases in nearly all RNA viruses.  Thus C. elegans RNAi pathways that include RdRps may give hints about host cofactors that viral RdRp proteins may require.  The best candidates for proteins that act in the same pathway as RdRp genes are those with the same pattern of presence and absence across phylogeny.

The RNA dependent RNA polymerases (RdRp) siRNA-amplifying proteins also have a distinctive phylogenetic profile---these RNA replicases are very common in plants and many fungi, but has been lost in the vast majority of animals, except for nematodes, ticks, mites, and spiders, and a smattering of bivalves and corals.The phylogenetic profile below shows that the 700 aa RdRp domain of RRF-3, RRF-1, EGO-1 or RRF-2 find excellent Blastp matches in nearly all nematode and Fungi and plants but not in the Saccharomycetes, which we know have jettisoned their RNAi pathway (blue intensity proportional to a high BlastP score in those species; white denotes no blastp homology).

 Surveillance of mitochondria and the ribosome for toxins and microbial attack

Our genetic analysis of how C. elegans surveils its core cellular components has revealed unexpected coupling to innate immunity and aging. This genetic analysis explains why, for example, the translation inhibitor rapamycin is anti-aging—it triggers a natural toxin surveillance system that is coupled to defense and aging programs. We continue to focus on C. elegans for genetic discovery. C. elegans molecular genetics, screening after random mutagenesis for mutant phenotypes and deducing the molecular defect by genome sequencing dozens of mutants, has become about 100x less costly per gene discovery since the development of low cost full genome sequencing. The genes we study have human orthologues, and are likely to function in an ancient conserved pathways for detection of microbial assaults and control of the aging process. This surveillance system may explain why women live longer than men and why they have dramatically higher frequencies of autoimmune disorders: our hypothesis is that this system of toxin surveillance and defense is more active in women, most likely as a fetal defense. Human variation in such defense programs could be the genetic dowry from survivors of past viral and bacterial pandemics. Our studies of how the microbial flora subvert these pathways may reveal how the microbiome may influence human longevity and the dramatic increase in viral vulnerability of the elderly, as starkly revealed by Covid19.

 The Search for Extraterrestrial Genomes (SETG) Project

Over the past 25 years, we have been developing a life detection and analysis instrument that can isolate, detect, and sequence nucleic acids on the surface or in orbit of another planet or moon. Low temperature meteoritic exchange between Mars and Earth has been documented by Martian meteorites.  If life on Mars uses the same molecules of life on Earth, then the sensitive tools of DNA analysis can be marshalled to detect life on Mars.  Gary Ruvkun and Mike Finney began to work on PCR amplification of metagenomic samples with the 16S gene primers in 1993.   In 2001, we connected with Maria Zuber, an eminent MIT planetary scientist to begin a 20 year collaboration.  Tom Isenbarger and Chris Carr joined the project, as did a series of MIT undergraduates and graduate students.  Our SETG instrument has now morphed to an Oxford Nanopore-like sequencer that will enable direct sequencing from double stranded DNA in a less than 100 gram package with minimal sample preparation. We predict that any Martian biota will be deeply branching in the phylogenetic tree of Earth DNA sequences. This phylogenetic analysis of any DNA detected on another body is central to ruling out contamination of DNA from Earth. 

A core of about 500 highly conserved protein and RNA components are common to all current life on Earth.  This core of genes had already evolved before in the last common ancestor to all life on Earth, LUCA, 3.5 to 4 billion years ago.  LUCA had evolved (or arrived) within a few hundred million years of the cooling of the Earth.   One hypothesis for why we find a complex LUCA bacteria very soon after the Earth became habitable is that LUCA only had to be naturally selected for the capacity to grow and reproduce from a complex meteoritic inoculum of organisms from outside the Solar System.  Within 50 light years of Earth there are about 50 star systems.  Many of these stars have exoplanets, some of them habitable, based on radiation levels, solar radiation and light levels, and predicted temperatures.  The orbital disruptions by large planet migrations are excellent engines for spreading life between stars: the ejection from stellar orbit of a habitable planet analogous to Earth by such a migrating hot Jupiter is a perfect vehicle for the insemination of life to the next stellar system with which such an ejected frozen planet might interact.   Migration of life across the galaxy gives the Tree of Life much more time to evolve.  The Milky Way is much older than the Solar System, 13.5 billion years vs 4.5 billion years. Another 9 billion years is a 100x time difference between the evolution of life in a few hundred million years from the primordial soup to genetic code and DNA world on the Earth.  And if the RNA world was on another planet more than 4 billion years ago, the geological and chemical history of the early Earth may have little relevance to the early steps in the evolution of the RNA world,. So there is some resistance to this idea from geologists and the geology-indoctrinated astrobiology and origin of life communities.  But they should embrace this idea: origin of life research becomes galactic in its explanatory power if it explores the precursor to life across the Milky Way rather than just life on the tiny blue dot of Earth.