A summary of 2023 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.

ADAR editing and the ERI-6/7 RNAi pathway silence endogenous viral elements and LTR retrotransposons (Fischer Ruvkun 2020)   Long double-stranded RNAs are a step in RNA virus replication.  Adenosine deaminases acting on double stranded RNA (ADARs) detect these RNA duplexes and edit the RNA sequence to destabilize the RNA duplexes.  A double mutant in the C. elegans ADAR genes, adr-1 and adr-2 grows normally, but if the animal also carries a mutation in the ERI-6/7 RNA  helicase or the ERGO-1 Argonaute protein that functions to silence newly acquired viruses, it is lethal (Reich Bass 2018; Fischer Ruvkun 2020).  The eri-6/7; adr-1; adr-2 triple mutant shows a dramatic upregulation of the RNAi machinery and the unfolded protein response, consistent with the secretory stresses associated with viral replication and secretion (Reich Bass 2018; Fischer Ruvkun 2020).  These animals are suffering from the toxicity of too much anti-viral RNA interference:  the lethality of the eri-6/7; adr-1; adr-2 triple mutant is rescued by inactivation of many RNAi factors such as mut-16, rde-1, nrde-3 (Reich Bass 2018; Fischer Ruvkun 2020).  A mutation in C. elegans drh-1, the C. elegans ortholog of the viral sensor protein RIG-I, also suppresses the lethality of the eri-6/7; adr-1; adr-2 triple mutant.   

 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.

Suppression of oxygen sensitivity of C. elegans electron transport chain mutants by mutations in a membrane protease or an RNA methyl transferase (research of Josh Meisel, a postdoc in both the Mootha and Ruvkun labs at MGH). Mutations in distinct Complex I subunit genes of the electron transport chain, nduf-7 and nduf-2/gas-1, are viable at atmospheric (20%) oxygen but are inviable at 50% oxygen, which does not inhibit wild type growth. By selecting for survival in 50% oxygen, from a population of more than 100,000 F2 progeny of nduf-7 or nduf-2/gas-1 animals after a random EMS mutagenesis, we identified dozens of mutations in a mitochondrial membrane protease gene and an RNA methyltransferase gene that suppress mutations in complex I subunits. Orthologues of these genes are located in the same operon in many species of bacteria, including E. coli, and are located in the same operon as the Complex I of the electron transport chain in some bacterial genera. Thus, the suppression of the oxygen sensitivity of two different C. elegans complex I mutations identified mutations in distinct classes of C. elegans nuclear genes that were located in the same operon, perhaps even in the bacteria endosymbiont 2 billion years ago that became the mitochondria. Our comparative genomics suggests a small set of candidate mRNAs that the RNA methylase may normally methylate, perhaps as regulated by oxygen tension. The phylogenetic profile of the RNA methylase is extraordinary and is highly correlated with genes that mediate the production of neurotransmitters such as dopamine. This RNA methylase may regulate the translation of these oxygen-requiring enzymes under particular oxygen tensions.

Induction of RNA interference by C. elegans mitochondrial dysfunction via the DRH-1/RIG-I homologue RNA helicase and the EOL-1/RNA decapping enzyme  Mao, K and Ruvkun. PLoS Biol. 2020. In mammals, mitochondrially-localized proteins such as MAVS, RIG-I, and MDA5 mediate antiviral responses. We found that mutations in many different C. elegans mitochondrial components robustly enhance RNA interference. These antiviral responses to mitochondrial dysfunction depend on the RIG-I homologue, the DRH-1 RNA helicase. Comparing the C. elegans transcriptional response of a mitochondrial mutant and infection with the Orsay RNA virus, we found a striking induction of multiple members of C. elegans pals- genes implicated in anti-viral and anti-pathogen response pathways, and the eol-1/DXO RNA decapping enzyme gene. eol-1 transcription is induction is DRH-1 dependent, and an eol-1 null mutation in strongly suppresses the antiviral RNAi response normally induced by mitochondrial dysfunction. A decrement in mitochondrial function is one of the most potent mechanisms to increase longevity in a variety of species. Mutations in eol-1 or drh-1 suppress the increase in longevity caused by mitochondrial dysfunction.  Thus, enhanced RNA interference and antiviral activity is a key output from mitochondria for anti-aging. The dramatic increase in frailty in human old age may reflect such an increase in viral vulnerability.

Protein sequence editing of C. elegans SKN-1A and its mammalian homologue Nrf1 by peptide:N-glycanase and its probable function in RNA virus immunity. Lehrbach and Ruvkun Cell. 2019, as well as unpublished Covid analysis.  Lehrbach Ruvkun. 2016. Elife. 2016. Lehrbach Ruvkun. Elife. 2019.  We isolated mutants that are no longer capable of activating expression proteasomal genes to ameliorate their proteasomal dysfunction; that is, these mutants are decoupled from normal proteasomal homeostatic control. We found that the PNG-1/NGLY1 N-deglyosylase is essential for the function of an isoform of the SKN-1 transcription factor that bears a transmembrane domain and is normally ER localized and degraded by the proteasome via ERAD. In addition, we discovered that the conserved DDI-1 protease is necessary for the proteolytic processing of SKN-1A. The DDI-1 protease is strongly induced by proteasome damage, is localized to the nucleus where it cleaves SKN-1. The short biological half-life of SKN-1A is a key sensor of proteasomal activity that in turn activates responses to proteasome dysfunction. Mammalian NGLY1 peptide:N-glycanase orthologous to C. elegans PNG-1 had been known for decades to remove an amine from the glycosylated asparagine as it deglycosylates the client protein to leave behind an aspartic acid residue in the deglycosylated client protein NxS/T glycosylation site (Suzuki 2002). But the glycosylation field was focused on the removal of glycosylation, not on the protein editing. To genetically test whether ER localization and N-glycosylation of SKN-1A followed by cytoplasmic deglycosylation and N to D editing is the actual mechanism of SKN-1A transcription factor activation, we changed the SKN-1A genome sequence to aspartic acid codons at 4 asparagine codons located in N-glycosylation sites that showed a characteristic pattern of N to D substitution between divergent nematode species. These N to D genome edits activated SKN-1A for the induction of proteasome gene expression even in the absence of PNG-1 deglycosylation and deamidation. Constitutive activation of SKN-1A by genetic edits of four glycosylated asparagines to aspartate confers extreme resistance to proteasome inhibitors and effectively ‘cures’ a C. elegans model of Alzheimer’s disease. Amazingly, in the cancer genome dependency map, where the gene dependencies of hundreds of tumor cell types are compared, there is highly significant similarity of tumor types that are most dependent on NRF1(aka NFE2L1), NGLY1, or DDI2 gene activities. The tumors that most depend on the NGLY1, DDI2, and NRF1 pathway may for example be more aneuploid, and thus need greater proteasome homeostasis to adjust to the gene dosage challenges on protein complexes of aneuploidy. Mass spectroscopy also validated the deamidation of NRF1 N-glycoyslated asparagines and showed that the HLA system recognizes these protein edits (Mei 2020).

N-glycosylation is a common feature of viral envelope proteins; Covid-19 Spike protein has 22 N-glycosylation sites, some located in the region that binds to the ACE2 receptor and mediates viral entry into host cells (Zhang 2020). Using the pattern of N to D substitutions in NxS/T N-glycosylation sites in phylogenetic comparisons related coronaviruses, we found 57 D-substituted N-glycosylation sites in Covid-19 protein orthologues in other coronaviruses that are candidates for N to D editing, including 13 in the Spike protein. This phylogenetic analysis suggests that N to D editing of viral proteins by NGLY1. Some of these N to D sequence changes have been observed in mass spectroscopy of viral peptides, in many cases, bound to HLA. NGLY1 deficient patients show aberrantly increased antibody titers toward rubella and/or rubeola following vaccination, and their parents report few cold or flu infections (Lam 2017). An RNAi screen for human gene inactivations that confer immunity to Enterovirus 71, an RNA virus, identified NGLY1 as a gene activity needed for viral replication (Wu 2016).

 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.