Montgomery, Taiowa et al. (2021) International Worm Meeting "piRNAs prevent runaway amplification of siRNAs from ribosomal RNAs, histone mRNAs, and other coding gene mRNAs"

Vijayasarathy, Tarah, Reed, Kailee, Montgomery, Brooke, Montgomery, Taiowa

[International Worm Meeting, 2021]

Piwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals. Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In C. elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate low-level 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants. Despite hyperproduction of 22G-RNAs in piRNA mutants, the effects on histone and rRNA expression are modest and may have a negligible impact on germline development.

Lee DA et al. (2005) J Med Genet "Genetics of skin appendage neoplasms and related syndromes."

Schneiderman P, Grossman ME, Celebi JT, Lee DA

[J Med Genet, 2005]

In the past decade the molecular basis of many inherited syndromes has been unravelled. This article reviews the clinical and genetic aspects of inherited syndromes that are characterised by skin appendage neoplasms, including Cowden syndrome, Birt-Hogg-Dube syndrome, naevoid basal cell carcinoma syndrome, generalised basaloid follicular hamartoma syndrome, Bazex syndrome, Brooke-Spiegler syndrome, familial cylindromatosis, multiple familial trichoepitheliomas, and Muir-Torre syndrome.

SN Parrish et al. (1999) International C. elegans Meeting "Investigating the fates of trigger RNAs and target transcripts in RNA-mediated interference"

AZ Fire, SN Parrish

[International C. elegans Meeting, 1999]

We seek to explore the molecular mechanisms responsible for RNA-mediated genetic interference (RNAi). In nematodes, introduction of double-stranded RNA corresponding to a segment of an endogenous genetic locus can result in specific silencing of that locus, essentially producing a knock out phenotype [1]. To date, evidence indicates that this interference reflects a post-transcriptional mechanism, resulting in the loss of the endogenous transcript [2]. Only a few molecules of dsRNA are required per cell to mediate interference, suggesting either an amplification or catalytic aspect of the process [1]. To gain an understanding of the mechanism of RNAi, we are examining the fates of the two key players in this pathway, the endogenous target RNA and the dsRNA effector molecule. First, we are attempting to follow alterations in the endogenous transcripts after the introduction of dsRNA. As a start, we are trying to map possible cleavage events or potential chemical modifications through primer extension and RT PCR of the target transcript. In a complementary set of experiments, we are also examining potential changes in the dsRNA triggering molecule. Through the characterization of the target and effector RNA molecules, we hope to acquire some insight into the mechanism of RNA-triggered silencing. With this knowledge, in conjunction with genetic identification of components in the pathway, it may be possible to unravel the events and intermediates essential for RNAi. 1. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 2. Montgomery, Xu, and Fire. PNAS 95, 15502

Fire A et al. (1998) Nature "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans."

Fire A, Mello CC, Kostas SA, Montgomery MK, Driver SE, Xu SQ

[Nature, 1998]

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.AD - Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. fire@mail1.ciwemb.eduFAU - Fire, AAU - Fire AFAU - Xu, SAU - Xu SFAU - Montgomery, M KAU - Montgomery MKFAU - Kostas, S AAU - Kostas SAFAU - Driver, S EAU - Driver SEFAU - Mello, C CAU - Mello CCLA - engPT - Journal ArticleCY - ENGLANDTA - NatureJID - 0410462RN - 0 (Calmodulin-Binding Proteins)RN - 0 (Helminth Proteins)RN - 0 (Muscle Proteins)RN - 0 (RNA, Antisense)RN - 0 (RNA, Double-Stranded)RN - 0 (twitchin)SB - IM

William M Winston et al. (2001) International C. elegans Meeting "A Screen for Mutations that Enhance and Suppress Systemic RNAi"

Christina Molodowitch, William M Winston, Craig P Hunter

[International C. elegans Meeting, 2001]

One of the hallmarks of RNAi in C. elegans is the systemic effect: injecting gene specific dsRNA into one tissue interferes with the expression of that gene in other tissues (Fire, A. et. al, 1998). In order to elucidate the mechanisms of systemic RNAi, we have developed an assay that has allowed us to identify mutants that are specifically suppressed in their ability to execute systemic RNAi, but are still able to maintain cell autonomous RNAi. This assay has also been used to identify mutants that are apparently enhanced for RNAi. We have screened approximately 600,000 genomes in search of suppressor mutants and approximately 100,000 genomes for enhancer mutants. Towards our goal of identifying the genes necessary for systemic RNAi, we are placing the mutations into complementation groups, mapping representative mutants to linkage groups, and characterizing the gene and tissue specificity of the suppressor mutants. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 19;391(6669):806-11

A Fire et al. (1999) International C. elegans Meeting "Structural requirements for the trigger in RNA-mediated interference"

J Fleenor, S Parrish, A Fire

[International C. elegans Meeting, 1999]

This poster will describe in vivo interference activities for a series of modified double-stranded RNAs that are based on segments of the genes unc-22 and gfp . Double stranded RNAs have been shown to act as potent inducers of gene-specific molecular silencing in C. elegans [a]. This process apparently reflects a well conserved control mechanism: recent reports have confirmed the effectiveness of dsRNA-triggered interference mechanisms in a variety of additional species including plant, insect, and protozoan systems [b-e]. By characterizing structural requirements (on the triggering side) for these two well defined segments in C. elegans , we hope to illuminate general features of the interference mechanism. Our experiments involve a variety of manipulations to produce dsRNAs with sequence or chemical alterations, followed by injection into C. elegans and assays for genetic interference. The manipulations are designed to address the following questions: 1. How precise and extensive are requirements for homology with the target gene? 2. What features distinguish the incoming RNA as "foreign"? 3. What chemical groups on the incoming RNA participate in interference? 4. Do the incoming sense and antisense strands have distinct roles in triggering interference? a. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 b. Waterhouse, Graham, Wang, PNAS 95, 13959 c. Ngô, Tschudi, Gull, Ullu, PNAS 95, 14687 d. Kennerdell and Carthew, Cell 95, 1017 e. Misquitta and Paterson, PNAS 96, 1451

Lisa Timmons et al. (1999) International C. elegans Meeting "On the Systemic Nature of dsRNA-mediated Genetic Interference in C. elegans"

Lisa Timmons, Andrew Fire, Jamie Fleenor

[International C. elegans Meeting, 1999]

We are investigating the mechanisms by which double-stranded RNA produces a systemic interference response. The ability of an exogenous dsRNA to interfere with the function of an endogenous gene has been described[1]. This was first observed in the progeny of animals whose germlines had been injected with dsRNA. Subsequent observations demonstrated the existance of a mechanism by which dsRNA injected into the body cavity of an adult (in which few somatic cell divisions occur) could affect the entire animal. In addition, C. elegans can respond in a gene-specific manner to dsRNA encountered in the environment. In particular, an RNAi effect can be observed when worms eat dsRNA expressed in bacteria[2]. This ability of cells outside the gut to be affected by dsRNA administered throught the gut has led us to the question of how dsRNA or some effect mediated by dsRNA spreads from the site of initial contact throughout the animal. We are currently investigating the extent of the "spreading effect" and are using genetic screens to identify genes that might mediate spreading. A second question related to systemic interference that we are addressing is that of differential tissue susceptibility of RNAi. We and other investigators have noted that some cells in the nervous system appear refractory to RNAi[3]. We are currently investigating the cellular pattern of this refractibility in the nervous system and are using genetic screens to identify components that enable neurons to become refractile to RNAi. [1] Fire, A., Xu, S.-Q., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Nature. 391: 806-811. [2] Timmons, L., and A. Fire. (1998) Nature. Oct 29: 395(6705):854.[3] Fire, A., and J. Fleenor. (1998). Worm Breeder's Gazette 15(3): 8.

Mary K Montgomery (2003) International Worm Meeting "Expanding the definition of "good science": incorporating discussion of ethics and public policy into the standard science curriculum"

Mary K Montgomery

[International Worm Meeting, 2003]

The typical introductory genetics course will cover many key topics, ranging from Mendelian to molecular to population genetics. In addition to teaching our students how to do genetics, through problem-solving, testing, and laboratory exercises, we may also want to help our students place the practice of genetics in a social, historical, and ethical context. I have had students in my introductory genetics courses and advanced molecular genetic and developmental courses read several papers and book chapters beyond the textbook and primary literature, including writings by S. J. Gould, J. Beckwith, and P. Kitcher. Students then write thesis-governed essays on topics ranging from eugenics, to genetically modified food crops, to human embryonic stem cell research. Students have found these assignments very engaging, as it allows them to connect what they are learning in the classroom to the world at large. Nonetheless, some students struggle with fully analyzing the pros and cons of an issue and may produce weak thesis statements (at best). In an effort to get students to more fully engage the material and to think about both the philosophical and practical aspects of modern genetic research, I now have them work in groups of 4 to 6 students, who form an "advisory panel" modeled after the (now defunct) National Bioethics Advisory Commission, to produce a public policy report, which includes an executive summary statement with specific regulatory recommendations. Because students view this type of assignment as a form of public scholarship that will be shared with their classmates and the larger campus community, they are motivated to dig deeper into the issues, to fully debate them, and to eventually reach consensus in order to draft a workable policy. By devoting two or three class periods per semester to these types of activities, we can encourage our students to develop into not only good scientists, but thoughtful engaged citizens. Samples of policy reports will be made available at the meeting; links to the reports will also be available online in May 2003 at www.macalester.edu/~montgomery/courses.html

DP Morse et al. (1999) International C. elegans Meeting "RNA interference depends on genes required for mRNA surveillance"

SE Mango, DP Morse, ME Domeier, M Portereiko, BL Bass

[International C. elegans Meeting, 1999]

Double-stranded RNA (dsRNA) injected into C. elegans inhibits expression of genes homologous to the injected RNA 1,2 . This phenomenon, called RNA interference (RNAi) in worms, may be related to homology-dependent gene silencing observed in other organisms. Seven smg genes are known to be involved in the degradation of aberrant transcripts, a process known as mRNA surveillance 3 . Since there is evidence that RNAi works through mRNA degradation 2 , we tested whether the smg pathway is required for RNAi. We observed that worms mutant in particular smg genes recovered rapidly from the effects of RNAi. In wild-type worms, the paralyzed phenotype resulting from injection of unc-54 dsRNA persisted throughout the lifetime of the affected progeny ( unc-54 encodes a myosin heavy chain). The progeny of smg-2 worms injected with unc-54 dsRNA displayed the paralyzed phenotype at early time points similar to what has been observed previously 2 . However, the smg-2 animals showed dramatically improved movement as they aged. Interestingly, while smg-3,4,5 & 6 worms also recovered from RNAi to varying extents, smg-1 animals did not show this pattern of recovery and remained paralyzed. Quantitative RT-PCR showed that unc-54 mRNA levels correlated well with the observed phenotypic patterns. We are currently targeting other genes by RNAi to test the generality of these observations. In summary, our data has (1) identified the first trans-acting factors required for RNAi; (2) linked the phenomenon of RNAi to the process of mRNA surveillance; (3) revealed phenotypic distinctions among the smg genes. One possible interpretation of our results is that RNAi proceeds through a two-step mechanism: mRNA is initially degraded in a smg -independent step and low RNA levels are maintained by a process that depends on a subset of smg genes. 1. Guo & Kemphues Cell 81, 611 2. Fire et al. Nature 391, 806; Montgomery & Fire PNAS 95, 15502. 3. Hodgkin et al. Genetics 123, 301; Pulak & Anderson Genes Dev 7, 1885; Cali et al. Genetics 151, 605.

Sonnhammer ELL et al. (1998) European Worm Meeting "Bioinformatics driven functional genomics: a multifront approach to understand complex pathways with a "simple" little worm"

Vaz Gomes A, Sonnhammer ELL, Krause MW, Schrimpf S, Wahlestedt C

[European Worm Meeting, 1998]

C. elegans has been a useful model organism for developmental studies because of its ease of culture, simple anatomy, and available genetics. These attractive features are now be augmented by a wealth of molecular data from a multitude of nematode research labs as well as data provided by the completion of the full genome sequencing project. Already, the genomic information is allowing accurate detection of C.elegans orthologues to various genes relevant in human diseases or other conserved pathways (Mushegian et al., 1997). The time is right to take a bioinformatics driven approach to functional studies. The combination of the complete genomic sequence, bioinformatics, and an experimentally facile organism makes C. elegans an excellent system in which to dissect complex signaling pathways. A large body of signal-transduction pathways involving seven-transmembrane G-protein coupled receptors mediate responses to different types of chemicals like odorants, neurotransmitters, hormones, and also to more !physical! stimuli, like osmotic pressure, temperature, pressure, etc. Many members of these types of pathways have been studied in some detail in C. elegans while other potential candidates have so far only been identified !in silico! (Sonnhammer & Durbin, 1997). Clearly, the nematode has been, and will continue to be, helpful in identifying potential roles for these factors in regulating behaviour and responses to environmental cues, or in crucial developmental processes. The emergence of RNA-mediated interference (RNAi) (Fire et al., 1998) provides a powerful technique that will facilitate the identification of phenotypes associated with the elimination of single or combinations of multiple signalling factors. To improve biochemical analysis in the worm, we aim to apply the new generation of protein 2D-gels analysis systems. We expect this to become a powerful tool (e.g. Bini et al., 1997), for example revealing mutation effects and more accurate proteomics. This is especially important for genes encoding transcription factors, where changes of the expression pattern might be detected most easily at the protein level. For such studies the main requirement is non-lethality, health and fertility of the mutation, so that enough material can be obtained for the analysis. REFERENCES Bini, L., Heid, H., Liberatori, S., Geier, G., Pallini, V. & Zwilling, R. (1997) Electrophoresis18, 557-562 Fire, A., Xu, SQ., Montgomery, M.K., Kostas, S., Driver, S.E. & Mello, C. (1998) Nature 391, 806-811 Mushegian, A.R., Bassett, D.E.Jr, Boguski, M.S., Bork, P. & Koonin, E.V. (1997) PNAS 94, 5831-5836 Sonnhammer, E. & Durbin, R. (1997) Genomics 46, 200-216