Mello, Danielle et al. (2021) International Worm Meeting "Rotenone modulates the Caenorhabditis elegans immunometabolism and pathogen susceptibility"

Bijwadia, Shefali, Bergemann, Christina, Caldwell, Alexis, Meyer, Joel, Baugh, Ryan, Chitrakar, Rojin, Fisher, Kinsey, Mello, Danielle, Wang, Yang

[International Worm Meeting, 2021]

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Many pesticides target mitochondrial function, including the well-characterized complex I inhibitor, rotenone (Rot). Exposures to these mitotoxicants can pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. Our hypothesis is that Rot can disrupt C. elegans immunometabolism and consequently alter pathogen survival. C. elegans eggs were exposed to Rot (0.5 microM) or vehicle (Ctrl - 0.125 uM DMSO) in liquid and harvested once they reached the L4 larval stage (which was ~24h later for the Rot treatment, as it caused growth delay). Inhibition of mitochondrial respiration by Rot was confirmed by measuring the worm oxygen consumption rate. To explore pathways that were modulated by Rot, we performed a transcriptomic analysis and found 179 differentially expressed genes. WormCat analysis revealed that the two major broad enriched categories were stress response -which was mostly represented by pathogen response and detoxification genes- and metabolism -which was mostly represented by lipid and mitochondrial metabolism genes. Next, Ctrl and Rot-exposed worms were depurated for 48h, and further exposed to Pseudomonas aeruginosa (PA14), and Salmonella enterica (SL1344). Rot-exposed worms were more resistant to SL1344 but more susceptible to PA14. The mitochondrial unfolded protein response (mitoUPR) is a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. Rot activated the mitoUPR pathway, which was evidenced by increased hsp-6:GFP expression. Activation was also observed after 24h of exposure to PA14 and SL1344, however, Ctrl PA14-infected worms displayed lower hsp-6:GFP expression, and Rot rescued its expression only to the level of Ctrl OP50-raised worms. Thus, mitoUPR activation could be involved in the increased resistance to SL1344 but the level of activation in PA14 worms might not have been sufficient to promote resistance. By further exploring our transcriptomic dataset using WormExp and "module-weighted annotations" analysis tools, we identified genes that are known to confer resistance to PA14 that were downregulated by the Rot exposure, including HIF-dependent genes, which may underlie the increased susceptibility to PA14. Together, these results demonstrate that the mitotoxicant rotenone can modulate important pathways associated to the C. elegans immunometabolism and alter pathogen resistance.

Mello C (2007) Cell Death Differ "Return to the RNAi world: rethinking gene expression and evolution."

Mello C

[Cell Death Differ, 2007]

Thanks to the Nobel Foundation for permission to publish this Lecture (Copyright((c)) The Nobel Foundation 2006). Here we report the transcript of the lecture delivered by Professor Craig C Mello at the Nobel Prize ceremony. Professor Mello vividly describes the years of research that led to the discovery of RNA interference and the molecular mechanisms that regulate this fundamental cellular process. The turning point of discoveries and the role played by all his colleagues and collaborators are described, making this a wonderful report of the adventure of research. The lecture explains in simple language the importance of this discovery that has added a great level of complexity to the way cells regulate protein levels; moreover, it points out the beauty and importance of Caenorhabditis elegans as a model organism and how the use of this model has greatly contributed to the advance of science. Finally, Professor Mello leaves us with a number of questions that his research has raised and that will require years of future research to be answered.Cell Death and Differentiation (2007) 14, 2013-2020; doi:10.1038/sj.cdd.4402252.

Papp A et al. (1995) International C. elegans Meeting "INVESTIGATION OF C. ELEGANS ELECTROPORATION"

Jahani A, Papp A

[International C. elegans Meeting, 1995]

C. elegans can be transformed efficiently with exogenous genetic material using the technique of microinjection (Mello et. al, 1991). Although microinjection is becoming easier and more economical through the use of devices like our $425 microINJECTOR System, the procedure relies on a microscope system and accessories costing between $10,000 and $30,000, as well as some dexterity and knowledge of worm anatomy.

Guedes SF et al. (1997) International C. elegans Meeting "The C. elegans MEX-1 protein is a P granule component that is required for the proper localization of other P granule components"

Hill RJ, Guedes SF, Priess JR

[International C. elegans Meeting, 1997]

In the nematode C. elegans, germ cells arise from early embryonic cells called germline blastomeres. Cytoplasmic structures called P granules are present in the fertilized egg and are segregated into each of the germline blastomeres during the first few cleavages of the embryo. Mutations in the maternally-expressed gene mex-1 disrupt the segregation of P granules, prevent the formation of germ cells, and cause inappropriate patterns of somatic cell differentiation (Mello et al., 1992; Schnabel et al., 1996). The mex-1 gene can encode for a protein, MEX-1, that contains two copies of a CCCH-type "finger" domain also found in the PIE-1 (Mello et al., 1996) and POS-1 (Hiroaki et al., this meeting) proteins of C. elegans (Guedes and Priess, 1997). MEX-1 (Guedes and Priess, 1997), PIE-1 (Mello et al., 1996) and POS-1 (Hill et al., this meeting) are expressed in the germline blastomeres, and are components of P granules. We show that MEX-1 is required to restrict PIE-1 expression and activity to the germline blastomeres during the early embryonic cleavages. We further show that MEX-1 is required for the localization of PIE-1 and POS-1 to the P granules but MEX-1 is not required for the P granule localization of MEX-3, GLH-1, and GLD-1, other P granules components.

Grishok A et al. (2000) Science "Genetic requirements for inheritance of RNAi in C. elegans."

Mello CC, Grishok A, Tabara H

[Science, 2000]

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.AD - Program in Molecular Medicine, Department of Cell Biology, University of Massachusetts Cancer Center, Two Biotech Suite 213, 373 Plantation Street, Worcester, MA 01605, USA.FAU - Grishok, AAU - Grishok AFAU - Tabara, HAU - Tabara HFAU - Mello, C CAU - Mello CCLA - engID - GM58800/GM/NIGMSPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (DNA Transposable Elements)RN - 0 (Helminth Proteins)RN - 0 (RNA, Double-Stranded)RN - 0 (RNA, Helminth)RN - 0 (rde1 protein)SB - IM

Huang, George et al. (2021) MicroPubl Biol

Rettmann, Aubrie, Waterland, Skye, Huang, George, DeMott, Ella, Sylvester, Melynda, Dickinson, Daniel J, Rhodes, Anita, Flynn, Abbey, Alicea, Persephone, Blanco, Sara, Ren, Cassie, Koh, Alex, Doonan, Ryan, de Jesus, Bailey, Meng, Carrie

[MicroPubl Biol, 2021]

The self-excising cassette (SEC) knock-in approach uses hygromycin selection and a visible roller phenotype to identify knock-ins, followed by a heat-shock induced excision of these visible markers to yield a seamless insertion of a fluorescent protein into the genome (Dickinson et al. 2015). Compared to protocols that utilize Cas9 protein and linear DNA repair templates (Paix et al. 2015; Dokshin et al. 2018; Ghanta and Mello 2020), the plasmid-based SEC approach employs a simpler screening strategy but requires more worms to be injected (Dickinson and Goldstein 2016).

Ghanta KS et al. (2021) STAR Protoc "Microinjection for precision genome editing in Caenorhabditis elegans."

Mello CC, Ghanta KS, Ishidate T

[STAR Protoc, 2021]

In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C.elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C.elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).

DeMott, Ella et al. (2021) MicroPubl Biol

Doonan, Ryan, DeMott, Ella, Dickinson, Daniel J

[MicroPubl Biol, 2021]

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.

Papp A et al. (1997) International C. elegans Meeting "WORM ZAPPING II: C. ELEGANS ELECTROPORATION?"

Lee JH, Farris K, Papp A

[International C. elegans Meeting, 1997]

C. elegans can be transformed efficiently with exogenous genetic material using the technique of microinjection (Mello et. al, 1991). Although microinjection has become straightforward through the use of devices like our $425 microINJECTOR System, the procedure relies on a microscope system and accessories costing between $10,000 and $30,000, as well as some dexterity and knowledge of worm anatomy. We previously reported evidence suggesting that exogenous DNA can be introduced into worms using The Cloning Gun, an inexpensive, hand-held, cordless, rechargeable electroporation device designed for high-efficiency transformation of Stationary-Phase E. coli, developed here at Tritech Research. The data were preliminary, and B-gal staining suggested gene expression in some patches of somatic cells in worms that could have either been P0 or F1; however, data were not available as to whether this transformation was heritable. Here we present data that electroporation can produce heritable transformation: F3 worms staining completely for B-gal. Attempts to replicate this result with rol-6 have produced only non-heritable rollers and dumpies. Because electroporation seems to be a low frequency transformation procedure, we are investigating other genetic markers that may allow for the recovery of individual transformants from a large population of non-transformed worms. Experiments with dye show that electroporation can readily permeablize both adults and eggs. Mello, C. et al. (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J. 10(12):3959-70.

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