Ishidate, Takao et al. (2021) International Worm Meeting "Proteolysis dependent gene silencing in C.elegans germline"

Makeyeva, Yekaterina, Ishidate, Takao, Shirayama, Masaki, Mello, Craig

[International Worm Meeting, 2021]

Animal germ cells employ small RNAs and Argonaute proteins to surveil transcripts and silence transposons and foreign genetic elements. Here we report that, in C.elegans germline, expression of a transgene encoding codon optimized Cas9 gene fused to GFP is silenced via small RNA dependent and small RNA independent mechanisms. Interestingly, small RNA-dependent silencing was initiated and small RNAs targeting Cas9::GFP sequence were generated even in the absence of prg-1 activity. Furthermore, disarming the small RNA pathway led to Cas9::GFP expression only in the most distal region of the gonad, suggesting that a second small RNA-independent silencing mechanism maintains the silencing throughout the pachytene region and in oocytes. A forward genetic screen revealed that inactivation of the ubiquitin-proteasome pathway causes full de-silencing of the CAS9::GFP throughout the germline, suggesting that the second silencing mechanism is mediated by protein degradation. Remarkably, insertion of virus derived T2A sequence that causes ribosome skipping, between Cas9 and GFP, lead to the expression of the downstream GFP moiety throughout the gonad. This observation suggests that the upstream Cas9 ORF is triggering protein degradation and that the mRNA is not degraded and can still encode downstream GFP. Thus ribosome subunits are likely not dissociated as in the RQC response to ribosome stalling. We propose that some modification within the upstream Cas9 mRNA signals for co-translational ubiquitylation and degradation of the peptide. Interestingly, a Cas9 transgene that contains multiple extended AT rich introns has been shown to escape this mode of silencing, pointing toward mRNA splicing as the triggering event and linking the phenomenon to the recently described intronless gene silencing. We are currently investigating the nature of the triggering signal and whether our findings extend to the regulation of endogenous targets.

Ishidate, Takao et al. (2009) International Worm Meeting "cdk-1(ne2257) suppressor screen and the developmental function of CDK-1/CYB-3 complex in polarity generation during early embryogenesis."

Shirayama, Masaki, Kim, Soyoung, Mello, Craig, Ishidate, Takao, Sharma, Rita

[International Worm Meeting, 2009]

We have isolated a rare temperature-sensitive allele of cdk-1(ne2257) that does not affect cell cycle progression but instead causes very specific developmental defects that appear to stem from a failure to degrade the CCCH-type zinc finger proteins OMA-1 and OMA-2 prior to the onset of the first mitosis. Persistence of the OMA-1/2 proteins leads to defects in polarity generation and in the expression of other developmental regulators. OMA-1/2 degradation depends on direct phosphorylations by a set of conserved kinases, including MBK-2, KIN-19 and GSK-3. However, although cdk-1 and cyclin B3 lesions both lead to a dramatic stabilization of OMA-1/2 proteins, their role in promoting OMA-1/2 degradation is not clear since unlike the other kinases, CDK-1/CYB-3 does not phosphorylate the OMA-1/2 proteins directly. In order to study the developmental function of the CDK-1/CYB-3 complex in generating embryonic polarity, we have undertaken a suppressor screen, looking for mutants that rescue cdk-1(ne2257) embryonic lethality at restrictive temperature. From this powerful screen we have isolated multiple rare suppressing mutations including reverting mutations in cdk-1 that restore the altered amino acid to wild-type, compensatory mutations in cyb-3 that accommodate the altered cdk-1-mutant interface, as well apparently three novel suppressors that map to LGI (2 alleles), LGII (6 alleles) and LGIV (1 allele). As expected, the cdk-1 and cyb-3 suppressor mutations rescue the OMA-1 degradation defect seen in cdk-1(ne2257). However, in contrast the novel suppressors on LGI, II and IV all continue to exhibit persistence of OMA-1, suggesting that they function downstream of the OMA proteins. We are currently attempting to clone these suppressors using a deep sequencing approach. Characterizing these suppressors will likely shed light on the role of the cell cycle machinery in coupling the onset of mitosis to polarity generation during C.elegans early embryogenesis.

Ishidate, Takao et al. (2009) International Worm Meeting "Regulation of WRM-1/b-catenin by the cell-cycle regulator CDK-1 during asymmetric cell division in C.elegans."

Ishidate, Takao, Sharma, Rita, Kim, Soyoung, Mello, Craig, Shirayama, Masaki

[International Worm Meeting, 2009]

In the 4-cell stage C.elegans embryo, the b-catenin related protein WRM-1 responds to a polarizing signal that induces the EMS cell to divide asymmetrically and accumulates in the nucleus of posterior daughter E. WRM-1 localizes uniformly to the cell cortex in EMS during the interphase, however, at the onset of M-phase, it dissociates from the cortex proximal to the signaling cell P2 in a Wnt-signaling dependent manner. We noticed a conserved region in WRM-1 that contains potential phosphorylation sites for CDK-1 and GSK-3. Through in vitro studies we found that CDK-1 can phosphorylate WRM-1 directly at this site priming WRM-1 for subsequent phosphorylation by GSK-3. WRM-1 proteins with mutations at either the CDK-1 and GSK-3 sites failed to be released from the cortex and its downstream asymmetric nuclear accumulation was also diminished. In a rare temperature-sensitive mutant of cdk-1(ne2257) that exhibits a partial defect in Wnt signaling, WRM-1 dissociation from the cortex was found to be defective. Consistent with these findings, mutant CDK-1 isolated from the embryonic extract showed reduced kinase activity toward WRM-1 recombinant protein. To investigate the physiological significance of these observations in vivo, we have used mosSCI to introduce single-copy insertions of wild-type and mutant WRM-1 constructs. While our interpretations of these preliminary studies are still complicated by a lack of full rescue from the wild-type construct, we were encouraged to find that for each different construct, Western-blot analysis revealed that independent transgene lines exhibited very reproducible levels of expression. We were surprised, however, to find that the mutation of the CDK-1 site resulted in a significantly stronger rescue of the wrm-1 mutant strain, and also resulted in much higher levels of protein expression in each of the 2 lines examined. Similar evidence for increased activity from this mutant WRM-1(DCDK) construct was observed from a bombardment derived transgenic line. The GSK-3-site deletion construct did not exhibit this enhanced level of rescue and expression. These findings support the notion that WRM-1 is a direct regulatory target of CDK-1, and call into question the importance of its cortical localization during endoderm induction. We are currently trying to improve the transgene rescue utilizing different WRM-1 constructs. Through these studies, we would like to shed more light on the potential involvement of cell cycle regulator in developmental decisions.

Takao Ishidate et al. (2003) International Worm Meeting "A large-scale screen for temperaure sensitive embryonic lethal mutants."

Craig C Mello, Kuniaki Nakamura, Chris Trzepacz, Daniel R Brownell, Masaki Shirayama, Takao Ishidate, Kaming Pang

[International Worm Meeting, 2003]

Embryogenesis in C. elegans is a truly remarkable, clockwork-like, process that is readily amenable to genetic analysis. However, most methods for probing the function of genes required for embryogenesis provide only snapshots of the loss of function phenotypes. Temperature sensitive alleles, on the other hand, can provide insight into distinct structural and temporal activities of a protein. But how hard is the task of obtaining ts-alleles representing a substantial fraction of the genes required for embryonic viability? To address this question, and to find interesting and useful new mutants, we have undertaken a very large screen for ts-embryonic lethal mutants. (see 2001 abstract 1083). To date we have collected over 4,000 ts embryonic lethal strains. The strains fall into five broad classes according to their embryonic-lethal phenotype (morphogenesis defect (40%), differentiation defect (20%), cell division defect (20%), cell fate change (2%) and variable (18%). Many of these strains also exhibit larval defects including lethality, sterility and mobility phenotypes. To gauge the depth of our collection, we have mapped 26 Mom/Wnt-signaling-endoderm defective mutants in our collection. Of 12 known pathway genes identified in previous forward and reverse genetic screens, our ts alleles identify 4 genes; lit-1/Nemo kinase (13 alleles), mom-2/Wnt (8 alleles), mom-4/Tak1 kinase (4 alleles) and wrm-1/-catenin (1 allele). Notably, alleles in these genes appear to arise at different rates. Lesions in the two kinases are common and occur primarily in the kinase domain. Lesions in the secreted Wnt protein are also common and tend to alter conserved cysteine residues. The single allele of WRM-1/-catenin alters a conserved leucine in one of the armadillo motifs. The reasons for the bias in allele frequency are unclear, for example alleles of another kinase in this pathway, GSK-3, were not identified in our screen to date. From these studies it is clear that most essential embryonic processes that involve multiple gene products are likely to be defined by one or more alleles in our collection. As for identifying all of the genes in a pathway such as the Wnt-pathway, it would seem that a collection on the order of at least several times the size of the current collection will be necessary. Because the collection of these mutants is labor intensive, it might be valuable at some time in the future to begin a project to systematically map the mutants or to disseminate the unmapped mutants to other interested parties. Ideas on how to do this will be welcomed.

Takao Ishidate et al. (2002) Worm Breeder's Gazette "A method of growing large synchronous population of C.elegans using chicken eggs."

Takao Ishidate, Craig Mello

[Worm Breeder's Gazette, 2002]

It is sometimes required to prepare a large synchronously growing population of worms for various biochemical or genetic assays. Use of chicken eggs to grow gram quantity of worms has been described (D.Baillie and R.Rosenbluth; WBG2(1)). We have found that by adding E.coli to blended chicken egg it is possible to grow a large population of worms synchronously on plates. In this method, freshly hatched L1 larvae are suspended in blended chicken egg solution supplemented with concentrated E.coli, and seeded onto standard NGM plates. Typically, 1 million worms can be grown synchronously on five, 150mm NGM plates. 1) Take one egg and break into 25ml of boiling water placed on magnetic stirrer. 2) Stir several minutes until yolk breaks. 3) Put the egg solution into Waring blender and blend for two minutes. 4) Transfer the homogenized egg solution to sterile tubes. (They can be frozen and stored at -80C.) 5) Mix egg solution and concentrated E.coli solution (OP-50 grown in Terrific Broth for one day is pelleted and then resuspended in equal volume of sterile M9). Usually we use the ratio of 1:9 = concentrated E.coli : egg solution. 6) Suspend 200,000 freshly hatched L1 larvae in 1~1.5ml of egg/E.coli mixture and inoculate onto 150mm NGM plate

Takao Ishidate et al. (2001) International C. elegans Meeting "Identification of embryonic lethal temperature sensitive mutants using a novel selection screen."

Chris Trzepacz, Kuniaki Nakamura, Craig C Mello, Takao Ishidate, Ka Ming Pang

[International C. elegans Meeting, 2001]

The short life cycle and rapid development of the nematode C. elegans make it an attractive organism for embryological studies. For example, genetic screens over the past decade have identified numerous factors involved in the control of cell polarity and cell fate in the early embryo. However, despite this progress many steps have yet to be defined by mutations. We know of at least two reasons for these missing steps, one of these is redundant genes that compensate for each other and thus preclude identification by single mutant events. The second reason involves individual genes that have multiple functions where disruption of an earlier function might preclude analysis of the developmental event of interest. There are no perfect solutions to these problems but at least one possible way to identify genes with multiple functions is to search for conditional alleles. Therefore we have recently initiated a screen for temperature sensitive (TS) embryonic lethal mutants. This screen utilizes the famous Jim Priess Egl mutant trick to trap mutant embryos inside their mother, as well as several novel selection methodologies to facilitate the identification new mutants. To facilitate genetic mapping of new alleles we have used the SNP rich C. elegans isolate from Hawaii as the starting genetic background. Following chemical mutagenesis of L4 animals, F1 and F2 progeny were reared at permissive temperature (15degC). A synchronous population of potentially homozygous F2 animals were shifted as late L4 stage animals to the restrictive temperature (25*C) and allowed to accumulate eggs. After 36 hours gravid F2 adults were returned to the permissive temperature and incubated for one more day to permit the fertilization and development of a few additional embryos if possible. These F2 animals were then subject to a brief hypochlorite treatment to kill all of the adults and any hatched larvae. Adults filled with unhatched eggs were then separated from other types of animals by sucrose flotation (we are not sure why this works but it does). Candidate TS mutants were then identified by looking for viable L1 larvae bracketing a collection of dead eggs. To date, we have screened the progeny of 6.4X106 mutagenized P0 animals and selected 2056 candidates. Approximately 16% (325) of these candidates actually harbor confirmed TS-embryonic lethal alleles. These TS-alleles have all been frozen and are categorized according to their restrictive temperature phenotypes. The majority of the mutants display a cell division and/or a tissue differentiation defect. Morphogenesis defects are also common, with the smallest class of mutants being those involved in cell fate decisions. Preliminary mapping and complementation tests suggest that at least a few of these define new mutant loci. There are also many new alleles of previously identified mutants. For example our gutless mutants include four lit-1 alleles, three mom-4 alleles, two par-2 alleles and two par-4 alleles. Our goal is to collect at least ten times the current number of ts mutants and then to assess whether or not it will be possible to saturate the C. elegans genome for TS-alleles. In the future, well mapped TS mutants from this and other similar screens conducted elsewhere, could provide a valuable resource to C. elegans researchers.

Takao Ishidate et al. (2007) International Worm Meeting "How does the CDK-1/CYB-3 complex couple the onset of mitosis and polarity generation in C.elegans early embryogenesis?"

Takao Ishidate, Craig Mello

[International Worm Meeting, 2007]

The CDK-1/CYB-3 complex is one of the major regulators of M-phase of the cell cycle. In early C. elegans embryos CDK-1/CYB-3 plays an essential role in generating cell polarity by promoting the timely proteolysis of the CCCH-type zinc finger proteins OMA-1 and 2. The degradation of the OMA proteins is also dependent on prior phosphorylations by a set of conserved kinases including MBK-2, KIN-19 and GSK-3, suggesting that OMA degradation is tightly regulated. CDK-1/CYB-3 appears to link OMA proteolysis to the onset of the first mitosis but how it does so is not yet clear since it does not appear to phosphorylate the OMA proteins directly. We have been analyzing rare alleles of cdk-1 that exhibit the abnormal stabilization of OMA proteins without any apparent cell cycle defects. These alleles alter conserved residues in the T-loop domain of the kinase, but do not affect cyclin binding or Histone H1 kinase activity, raising the possibility that these mutations abolish the ability of the kinase complex to recognize specific substrates relevant to OMA destruction. In order to find targets of CDK-1/CYB-3, we have been performing suppressor screens utilizing a temperature-sensitive allele of cdk-1(ne2257). The screen appears to be very powerful, generating rare and specific mutations. For example, from F1 dominant screens we have isolated 2 alleles of cyb-3 (ne4276 and ne4277) that alter residues within the N-terminal a-helix of the cyclin that directly contact the T-loop of of activated CDK-1. Remarkably, according to the crystal structure of activated human Cdk2/Cyclin A complex, the Isoleucine-114 to Valine change in CYB-3 (ne4276) compensates the subtle structural hindrance created by the original Isoleucine-173 to Phenylalanine change in CDK-1 (ne2257). So far 19 suppressors that do not have cyb-3 mutations have been identified. Nine of these map to LGIII near CDK-1 and so far five of these have been confirmed as intragenic reversions in cdk-1. Of the remaining 10 lines, 8 are confirmed to be neither cdk-1 nor cyb-3. We are currently mapping these extrageneic suppressors. We are also attempting to identify CDK-1/CYB-3 targets by biochemical means. Through these experiments we would like to better understand the intricate interplay between cell cycle machinery and development. cdk-1cyb-3<font face=symbol>a</font>.

Metali, Redi et al. (2019) International Worm Meeting "The germline KH protein, TOFU-7, promotes maternal-effect fertility and the localization of the Piwi Argonaute, PRG-1 to germline nuage."

Metali, Redi, Ishidate, Takao, Mello, Craig

[International Worm Meeting, 2019]

piRNAs in C. elegans are an abundant and diverse class of small RNAs that interact with the PIWI ortholog PRG-1 to promote fertility and transcriptome surveillance. They are referred to as 21U-RNAs due to their bias for a 5U and a length of 21nt. Biogenesis of piRNAs is a multistep and poorly understood process. Previous findings have shown piRNA precursors are ~26nt long transcripts, 5? 7-methylguanylate capped, and transcribed by POL II starting exactly 2nt upstream of the 5U. Processing of these precursors into mature piRNAs is thought to involve 5? decapping and trimming to expose the 5? U, likely followed by loading and then 3? end trimming by the exonuclease PARN-1. In order to find factors that are involved in decapping or 5 trimming, we tested several candidates. Our findings suggest that a previously reported piRNA biogenesis factor, TOFU-7 (Goh et al, 2014), may be involved in regulating piRISC stability. TOFU-7 has two KH domains suggesting that it may function as an RNA binding protein. In order to understand the putative functions of those domains, we used CRISPR to introduce mutations targeting each KH domain. Null alleles showed a strict maternal effect sterile phenotype, suggesting that TOFU-7 is playing additional roles independent of piRNA processing. In frame disruption of the first KH domain resulted in a phenotype similar to the null allele. Interestingly, however, deletions removing portions of the second KH domain were homozygous viable, with markedly reduced and variable brood sizes, suggesting that the two KH domains may have separate functions. The viable KH2-mutant alleles exhibit a largely wild-type germline development including production of normal numbers of mitotic and meiotic germ nuclei, and of both sperm and oocytes. The general P-granule marker PGL-1 exhibits a wild-type perinuclear distribution within the gonad. In contrast, however, PRG-1 localization in nuage is nearly completely abolished, instead distributing broadly within the gonad cytoplasm. Strikingly, PRG-1 also accumulates in the nuclei of late stage oocytes in these mutants. Together, these findings suggest that in addition to functions necessary for germline development, TOFU-7 may promote the proper localization of PRG-1. Further work is required to understand how TOFU-7 and PRG-1 interact to promote small-RNA silencing pathways in the germline and how the KH2 TOFU-7 alleles influence PRG-1 localization.

Durning, Daniel et al. (2021) International Worm Meeting "PRG-1 and ZNFX-1 act in parallel to regulate small RNA-mediated transgenerational epigenetic inheritance"

Mello, Craig, Ozturk, Ahmet, Durning, Daniel, Ishidate, Takao

[International Worm Meeting, 2021]

The PIWI-interacting RNA (piRNA) pathway in Caenorhabditis elegans engages thousands of endogenously encoded piRNAs within perinuclear nuage (P-granules) to scan RNAs exiting the nucleus. Sufficient base-pairing between a piRNA and a target RNA elicits a silencing response that is maintained transgenerationally by worm-specific Argonaute (WAGO) pathways following recruitment of RNA-dependent RNA polymerases (RdRPs) which synthesize 22 nt RNA species with a bias for a 5' guanosine (22G-RNAs). We previously described ZNFX-1 as a factor involved in piRNA-triggered RNA-induced epigenetic silencing (RNAe); ZNFX-1 was also found to engage the CSR-1 pathway which targets endogenous genes to protect them from silencing. Our study identified ZNFX-1 as being involved in positioning of RdRPs along small RNA-targeted transcripts to promote uniform 22G-RNA production to prevent bias for 5' targeting of mRNAs. Here we report that ZNFX-1 acts in parallel with PRG-1 to affect epigenetic inheritance of signals for both CSR-1 and WAGO target genes; small RNA sequencing shows us that 22G-RNAs targeting WAGO-target genes are strongly reduced in prg-1;znfx-1 double mutants relative to either single mutant or wild-type, and the double mutant sees increased expression of transposases for transposons Tc1 and Tc3 as shown by quantitative PCR (qPCR). Some small RNA targets whose targeting 22G-RNAs are depleted in a prg-1 single mutant see increased small RNA production in prg-1;znfx-1, which may be the result of these genes becoming targets for CSR-1 rather than WAGOs. Furthermore, we see that unlike znfx-1 single mutants which are defective in RNAi inheritance, prg-1;znfx-1 double mutants can inherit RNAi, consistent with the idea that loss of endogenous small RNAs frees up small RNA machinery to respond to an exogenous RNAi trigger, also suggesting that PRG-1 activity negatively regulates, and ZNFX-1 function is dispensable for, RNAi inheritance. Moving forward, we will immunoprecipitate Argonautes in the CSR and WAGO pathways and sequence the small RNA populations being loaded into these Argonautes to better understand how loss of PRG-1 and ZNFX-1 activity affects endogenous small RNA pathways, and we will sequence small RNAs in animals grown on RNAi to develop a clearer picture of how these factors act in RNAi inheritance. We will also use DNA and RNA fluorescence in-situ hybridization (FISH) to look at spatial regulation of RNAs undergoing desilencing in these mutants.

Elewa, Ahmed et al. (2013) International Worm Meeting "Suppressors of pos-1 identify a novel function for GLP-1 and new players, gld-3, cyclin E and spos-1, involved in endoderm specification."

Mello, Craig, Ishidate, Takao, Vergara, Sandra, Elewa, Ahmed, Shirayama, Masaki

[International Worm Meeting, 2013]

Maternal mRNAs program much of early embryogenesis, however the mechanisms that control their remain mysterious. POS-1 is a Cys3His zinc finger protein that regulates the translation of maternal RNAs. In pos-1 mutants the fates of the P2 and EMS blastomeres are misspecified leading to incomplete pharyngeal development and an absence of both endodermal cells and germ cells and . In addition, pos-1 mutants exhibit a defect in the Notch-signaling-dependent specification of the ABp blastomere. While the ABp defects in pos-1 mutants are correlated with missexpression of maternal mRNAs encoding the Notch signaling components GLP-1 and APX-1, nothing is known at present about what mechanisms underlie the pos-1 defects in EMS and P2 development. To begin to address this question we undertook RNAi and genetic screens for factors that restore endoderm specification in pos-1 mutants. The RNAi screen identified the KH domain gene (gld-3), Cyclin E (cye-1) and the novel gene F32D1.6 (which we have named "suppressor of pos-1" spos-1). Forward mutagenesis screens identified alleles of two genes gld-3 and glp-1. In each case suppression results in restored endoderm and pharyngeal-mesoderm differentiation, but does not restore germ-line specification or Notch signaling. The glp-1(ne4298) lesion alters a conserved amino acid in the 4th swi6 motif resulting in a strong temperature sensitive glp-1 loss of function phenotype. Surprisingly, the temperature-sensitive period (TSP) for glp-1 suppression of pos-1 occurs prior to fertilization, indicating that glp-1(ne4298) alters a maternal function that influences endoderm specification several hours later during early embryogenesis. The spos-1 gene has no obvious conserved motifs. However, spos-1 single mutants exhibit a maternal effect lethal phenotype with properly specified and well-differentiated tissues. We are investigating whether POS-1, GLP-1 and other pos-1 suppressors regulate spos-1 expression. We will present our characterization of SPOS-1 and ongoing efforts to understand the surprisingly complex nature of endoderm specification.