Tyc KM et al. (2017) Dev Cell "The Conserved Intron Binding Protein EMB-4 Plays Differential Roles in Germline Small RNA Pathways of C.elegans."

Tyc KM, Nabih A, Sobotka JA, Claycomb JM, Wedeles CJ, Wu MZ

[Dev Cell, 2017]

Proper regulation of the germline transcriptome is essential for fertility. In C.elegans, germline homeostasis hinges on a complex repertoire of both silencing and activating small RNA pathways, along with RNA processing. However, our understanding of how fundamental RNA processing steps intersect with small RNA machineries in the germline remains limited. Here, we link the conserved intron binding protein, EMB-4/AQR/IBP160, to the CSR-1 and HRDE-1 nuclear 22G-RNA pathways in the C.elegans germline. Loss of emb-4 leads to distinct alterations in CSR-1- versus HRDE-1-associated small RNA and mRNA transcriptomes. Our transcriptome-wide analysis shows that EMB-4 is enriched along pre-mRNAs of nearly 8,000 transcripts. While EMB-4 complexes are enriched for both intronic and exonic sequences of HRDE-1 targets, CSR-1 pathway targets are enriched for intronic, but not exonic, sequences. These data suggest that EMB-4 could contribute to a molecular signature that distinguishes the targets of these two germline small RNA pathways.

Claycomb, J.M. et al. (2017) International Worm Meeting "The conserved intron binding Protein EMB-4 plays differential roles in germline small RNA pathways."

Tyc, K.M., Wedeles, C.J., Claycomb, J.M., Wu, M.Z., Nabih, A., Sobotka, J.A.

[International Worm Meeting, 2017]

Proper regulation of the germline transcriptome is essential for the maintenance of fertility and survival of a species. In C. elegans, germline transcriptome homeostasis hinges on a complex repertoire of small RNA pathways that act in both activating and silencing capacities. Our understanding of how fundamental RNA processing steps, such as splicing, capping, nuclear export, and poly-adenylation intersect with these small RNA machineries in the germline remains relatively limited. Here, we link the conserved intron binding protein and splicing factor, EMB-4/AQR/IBP160 to two key 22G-RNA pathways in the C. elegans germline. EMB-4 associates with the Argonautes CSR-1 and HRDE-1, and is enriched at the genomic loci of CSR-1 and HRDE-1 target genes. Loss of emb-4 leads to distinct alterations in CSR-1 vs. HRDE-1 small RNA and mRNA transcriptomes. Our transcriptome-wide analysis shows that EMB-4 is enriched along pre-mRNAs of nearly 10,000 transcripts. For a subset of these genes, including mostly CSR-1 pathway targets, EMB-4 enriches for intronic, but not exonic, sequences. Notably, loss of emb-4 or the Argonautes with which it associates leads to similar defects in germline chromatin resetting in the primordial germ cells of embryos. Together these data point to EMB-4 as a factor that may help to enable the biogenesis of small RNAs and distinguish the targets of these two germline nuclear small RNA pathways in the worm. In turn, these activities are important for proper germline development and contribute to fertility over multiple generations.

Miller KG et al. (1995) International C. elegans Meeting "UNC-26: CHARACTERIZATION OF MUTANTS AND MOLECULAR GENETICS"

Miller KG, Rand JB

[International C. elegans Meeting, 1995]

Unc-26(md250) shows defects in both ACh synaptic transmission, as indicated by aldicarb resistance, and GABA synaptic transmission, as indicated by an expulsion defect. This suggests that the protein acts presynaptically. The primary defect is unknown but it does not seem to be a major disruption of the release mechanism itself because 1) mutants are not hypersensitive to levamisole, as is seen with several mutations known to inhibit neurotransmitter release, and 2) ACh levels per worm are only increased by 35%. The expulsion defect (Exp), defined as >10% failure, is seen in only 5 of the 18 alleles. Interestingly, some non-Exp alleles are just as severely impaired in pharyngeal pumping and movement as the Exp alleles, and 1 of the Exp alleles, e314, is a relatively mild eat/unc. This suggests the protein contains partially independent protein domains. To further define the role of unc-26 we are cloning the gene, which is tightly linked to the right part of the cosmid JC8. JC8 lies just left of a 165 kb gap. Using JC8 and a 2.7 kb rightward extension as probes, we detected 2 RFLPs among the 18 alleles. One is a complex RFLP in e314 recognized by JC8. The other is either a deletion or insertion in md250 detected with the 2.7 kb extension. The RFLP is not present in the parent strain of md250, RM25. Despite extensive screening, we have been unable to walk more than 2.7 kb into the gap, suggesting that this region may interfere with bacterial growth. We are currently trying to clone the 11.5 kb Hind III fragment carrying the RFLP directly from both N2 and unc-26 (md250) genomic DNA. Supported by an NRSA to K.M.

Chris Merritt et al. (2005) International Worm Meeting "A stem-loop in the 3UTR of nos-2 is required for translational repression in oocytes."

Pei-Lung Chen, Kuppuswamy Subramaniam, Chris Merritt, Ingrid D'Agostino, Geraldine Seydoux

[International Worm Meeting, 2005]

Nanos is a conserved regulator of germ cell differentiation. In many organisms, nanos RNA is maternally inherited and is translated only in the germ plasm, a specialized cytoplasm that segregates with the germline during early embryogenesis. How nanos translation is activated specifically in the germ plasm is poorly understood. We are investigating this question using the C. elegans nanos homolog nos-2. nos-2 encodes a maternal RNA that is enriched on P granules (germ plasm organelles) and is translated specifically in the germline founder cell, P4 , and its daughters the primordial germ cells Z2 and Z3. This expression depends on the nos-2 3'UTR. A GFP:Histone H2B:nos-2 3'UTR transgene (driven by the pie-1 promoter) is expressed only in P4 and Z2 and Z3. To begin to identify the transacting factors that act on the nos-2 3'UTR, we compared nos-2 3UTR sequences from C. elegans, C. briggsae and C. remanei. We noticed several conserved blocks including a conserved inverted repeat with the potential to form a stem loop. To test the significance of this structure, we created two mutations in the GFP:Histone H2B:nos-2 3'UTR transgene predicted to disrupt pairing in the stem (M1 and M2), and a double mutant (M1M2) predicted to restore pairing. We found that the M1 and M2 mutations lead to ectopic expression of the GFP:H2B reporter in oocytes. In contrast, the M1M2 double mutant was expressed in a pattern indistinguishable from wild-type (OFF in oocytes, ON in P4 and Z2 and Z3), consistent with restoration of the stem loop. We conclude that inhibition of nos-2 translation in oocytes depends on the formation of a stem-loop in the nos-2 3UTR. Interestingly translational inhibition of Nanos in Drosophila oocytes also depends on a stem loop (Forrest, K.M. et al. (2004). Development 131: 5849-5857).

Zhang L et al. (2010) Biology of the C. elegans Male, Madison, WI "Genetic Basis of Development and Mating Behavior Evolution in C. elegans"

Zhang L, Garcia L

[Biology of the C. elegans Male, Madison, WI, 2010]

A chemically defined axenic medium, C. elegans maintenance medium (CeMM), has been established for liquid culturing of C. elegans (Lu & Goetsch, 1993). Since organisms must coordinate behavioral and physiological response to changing environmental conditions, we are interested in how CeMM, as an environmental condition, affects development and mating behavior in C. elegans. Worms grown on CeMM plates take longer to develop and exhibit a prolonged reproductive period with a decreased brood size. For N2 animals, L1 to L4 larval development extends for seven days. Adult hermaphrodites would then lay eggs for approximately 10 days, which is similar to a previous report (Szewczyk et al., 2006). Compared with N2, CB4846, a Hawaiian strain, grows much slower and after 16 days, has fewer progeny (about 10 worms in the 1st generation). Interestingly, unlike N2, the 1st generation of CB4856 progeny developmentally arrests in L1, suggesting that the 1st generation of CB4856 could not utilize or synthesize some critical factors that N2 can. We have crossed N2 and CB4856, and obtained 200 recombinant inbred lines, which will be used to map the genes responsible for the viability difference between the two strains. We have also begun to study how CeMM affects reproductive behavior in the male. The male does not successfully copulate in CeMM. We found that a possible reason for reduced mating efficiency was that neuron-muscular circuits induced spicule protraction had a reduced responsive to acetycholine agonists such as arecoline. We are in the process of figuring out how genes control the development and mating behavior of N2 strain in CeMM, and what is the relationship between development and mating behavior in the experimental evolution history. To do this, we are screening for EMS generated N2 mutant hermaphrodites that grow faster and are more fertile in CeMM. We will serially select and then EMS-mutate hermaphrodites to gradually breed lines that become more adapted and fit for growth in CeMM, we will then ask if male mating, a behavior not directly selected for also adapts in the axenic medium. Lu N.C. & Goetsch K.M., 1993. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 39:303-311. Szewczyk N.J. et al., 2006. Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. J Exp Biol 209:4129-4139.

Zhang, Liusuo et al. (2011) International Worm Meeting "A recessive gene controls adaptation to a chemically defined medium in C. elegans."

Zhang, Liusuo, Garcia, L. Rene

[International Worm Meeting, 2011]

A chemically defined axenic medium, C. elegans maintenance medium (CeMM), has been established for culturing of C. elegans (Lu & Goetsch, 1993). Worms grown on CeMM plates take longer to develop and exhibit a prolonged reproductive period with a decreased brood size (Szewczyk et al., 2006). For N2 animals, about 10% of them develop into adults at day 8 after hatching, whereas the others develop into adults within the following 10 days. Compared to the lab-bred N2 strain, CB4856, a wild Hawaiian strain, takes greater than 16 days to reach adults. We are interested in determining genetic and molecular basis of developmental evolution that promotes animal cells to adapt to alternative nutritional environments. By screening known mutants which are possibly involved in feeding and growth, we observed that hermaphrodites contain mutations in fat-3, lev-11, ser-7, tph-1 and unc-73 grow much faster than N2 worms in CeMM (30-80% mutants reach adults between 5 and 7 days), while daf-2, daf-19, che-2, mdt-15, osm-9, unc-26, unc-32 and unc-42 mutants grow much slower or developmentally arrest in L1 (the majority could not develop into adults by two weeks). Through screening for EMS generated N2 mutant hermaphrodites that grow faster in CeMM, we identified the rg1402, rg1003 and rg804 alleles, which cause 90%, 90% and 75% of the worms to develop into adults on the synthetic medium by day 5, respectively. In addition, we have screened another 10 alleles which could develop to adults between day 5 and 6. Two alleles which cause the worms develop into adults at day 4 were identified through EMS mutagenesis of rg1003. We will serially select and then EMS-mutate hermaphrodites to gradually breed lines that become more adapted and fit for growth in CeMM. Outcrossing experiments demonstrated that enhanced adaptation to CeMM caused by rg1402, rg1003 and rg804 are inherited as a single-locus recessive trait. We are using classical mapping together with whole genome sequence of rg1003 to determine the molecular identity of the recessive allele. The characterization of the fast growth mutants in CeMM will greatly facilitate to systematically analyze the profound impact of nutrition on animal physiology, energy homeostasis and metabolism diseases. Lu N.C. & Goetsch K.M., 1993. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 39:303-311. Szewczyk N.J. et al., 2006. Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. J Exp Biol 209:4129-4139.