Donnelly, Bridget et al. (2019) International Worm Meeting "Interrogating the regulation and function of the mir-35 family of microRNAs in C. elegans."

McJunkin, Katherine, Donnelly, Bridget

[International Worm Meeting, 2019]

The mir-35 family of microRNAs are developmentally regulated in Caenorhabditis elegans and are maternally contributed as well as zygotically expressed in early embryos. Expression of the mir-35 family is essential for viability; complete loss of the mir-35 family results in lethality. The mir-35 family is tightly regulated at various stages during C. elegans development; mir-35 family is haploinsufficient for the regulation of sex determination indicating that the embryonic steady-state abundance is tightly controlled. In addition, mir-35 family abundance is developmentally regulated and is sharply decayed at the end of embryogenesis. Because of the tight regulation of the mir-35 family at steady-state and during development, this family of miRNAs is a strong candidate for studying the mechanisms of miRNA decay. The mir-35 family has two defining characteristics: the first is a shared, family-specific seed sequence, and second, mir-35 is preferentially loaded into the Argonaute protein, ALG-2. We are interested in exploring both of these characteristics to ascertain their roles in the stability or decay of the mir-35 family. To examine if the mir-35 family turnover is seed sequence-specific, we mutated the seed sequence of the mir-35 miRNA and monitored mir-35 levels past embryogenesis. We detected a perdurance of mir-35 levels past embryogenesis indicating that mir-35 family turnover is seed sequence-specific. Reciprocally, we are interrogating whether the 3' end of mir-35 plays a critical role in the stability or decay of the mir-35 family by mutating mir-35 outside of the seed region and examining the temporal expression of mir-35. Additionally, we seek to explore the role of ALG-2 in the stability of the mir-35 family. Strains containing tagged ALG-1 or ALG-2 in a background with the mir-35 seed mutants will be analyzed by immunoprecipitation followed by qPCR. These experiments will determine whether the delayed decay of mir-35 seed mutants is due to aberrant loading of the microRNA (e.g. loading into ALG-1). Overall, we aim to understand the regulation of mir-35 family abundance in early development and the role of the mir-35 family in C. elegans. By elucidating the mechanisms of the mir-35 family decay, this research will offer insight into the broader mechanisms regulating miRNA steady state abundance and turnover.

Huang, W. et al. (2019) International Worm Meeting "Features of histone H2A variant HIS-35 incorporation before and after fertilization."

Huang, W., Chu, D.S., Corpuz, C., Shorrock, M.

[International Worm Meeting, 2019]

To successfully pass down genetic material to the next generation, proper germ cell and embryo development is essential. These developmental processes are facilitated by proper gene regulation that is under the control of core histone proteins and their respective histone variants. By exchanging core histones with their variants, chromatin structure is remodeled to alter DNA accessibility to target genes. Previous research has identified 3 histone H2A variants namely, HTZ-1, HTAS-1, and HIS-35 in C. elegans.HTZ-1 is an evolutionary conserved H2A variant that is vital for development. Studies revealed parent derived HTZ-1 is removed from both maternal and paternal genome shortly after fertilization. This is in contrast to the sperm-specific H2A variant HTAS-1, which is retained in the paternal genome after fertilization. These results suggest how maternal and paternal-contributed H2A variants can be processed differently after fertilization. However, the role and processing of HIS-35 is unknown. Preliminary his-35 mutant studies revealed a 35% reduction in progeny when compared to the wildtype. In addition, following a GFP tagged his-35 strain has shown HIS-35 incorporation in spermatogenic, oogenic germline and fertilized embryos. These results suggested that HIS-35 can be involved in both fertility and embryogenesis. I hypothesize that HIS-35 regulate fertility associated genes during germ cell development. To assess HIS-35's role in fertility, we utilized a strain expressing HIS-35::GFP to examine HIS-35 incorporation throughout germline progression. Our results showed that HIS-35 colocalizes with H3K4me2, a mark of active transcription but not with H3K9me2, a repressive mark. This indicated that HIS-35 may play a role in regulating target genes needed for proper germ cell development. To assess HIS-35's role during embryogenesis, we tracked maternally derived (matHIS-35) and paternally derived HIS-35 (patHIS-35) separately during embryo development. Our results showed differences in HIS-35 incorporation between matHIS-35 and patHIS-35. Following 1 to 2 cell stage embryos, patHIS-35 was removed from the paternal genome shortly after fertilization. At the same time, matHIS-35 incorporates into the paternal genome and was retained in the maternal genome. Following 4 cell to late stage embryos, matHIS-35 incorporates into chromatin throughout embryogenesis. However, patHIS-35 was only detected starting at late stage embryo. These results suggested parent specific regulatory roles of HIS-35 post fertilization. This study provides further evidence that histone variants are removed and incorporated distinctly in the new embryo to maximize fertility.

McJunkin, Katherine et al. (2013) International Worm Meeting "The mir-35 family of microRNAs regulates hermaphrodite fecundity, male development, and genetically interacts with the sex determination pathway."

Ambros, Victor, McJunkin, Katherine

[International Worm Meeting, 2013]

The mir-35-42 family is one of three known essential microRNA families in C. elegans. Deletion of all eight microRNAs in the family results in embryonic lethality. To better understand the function of the mir-35 family, we have examined a viable strain in which seven of eight family members are deleted (mir-35-41), resulting in temperature-dependent embryonic lethality. First, we further characterized the mir-35-41 phenotype, and observed that mir-35-41 also exhibits a temperature-dependent defect in hermaphrodite fecundity, and males display abnormal tail morphology and reduced mating efficiency. We hypothesized that proteins that bind specifically to the mir-35-RNA-induced silencing complex (Wu, et al.) may be modulators of mir-35 family activity in the context of these phenotypes. Thus we examined genetic interactions between these candidate genes and the sensitized mir-35-41 background. In particular, inactivation of sup-26 strongly enhanced mir-35-41 lethality, suggesting that SUP-26 may act as a positive co-factor for the remaining mir-35 family member (mir-42). Paradoxically, sup-26 mRNA bears a conserved mir-35 family target site. If sup-26 is aberrantly up-regulated in mir-35 family mutants, then sup-26 inactivation should suppress the mir-35-41 phenotype. Indeed, a weak sup-26 allele partially suppresses the temperature-sensitive defect in fecundity of mir-35-41 (while enhancing embryonic lethality). Thus, sup-26 may have a dual role as a positive mir-35 family regulator and a mir-35 target gene. SUP-26 is an RNA-binding protein that plays a masculinizing role in both hermaphrodites and males through translational inhibition of tra-2 mRNA. tra-2 does not contain a conserved mir-35 binding site, and tra-2 loss of function does not suppress mir-35-41 embryonic lethality. However, in addition to sup-26(lf), other feminizing mutations, such as tra-2(gf) or fem-3(lf) also enhance lethality of mir-35-41. Our future work aims to elucidate whether SUP-26 indeed regulates the activity of mir-35-42 or other microRNA families, and to gain a clearer understanding of how the mir-35 family functionally interacts with the sex determination pathway.

Donnelly, Bridget et al. (2021) International Worm Meeting "Developmentally regulated microRNA decay of the mir-35 family is seed sequence dependent"

McJunkin, Katherine, Donnelly, Bridget

[International Worm Meeting, 2021]

The mir-35 family of microRNAs (miRNA) in Caenorhabditis elegans are maternally contributed as well as zygotically expressed in early embryos. Expression of the mir-35 family, which consists of 8 miRNAs, mir-35-42, is essential for viability; complete loss of the mir-35 family results in embryonic lethality. mir-35 family abundance is developmentally regulated and sharply decays at the end of embryogenesis. While there is much known about the biogenesis and functions of miRNAs, very little is known about the decay mechanisms of miRNAs. Because of this tight regulation of the mir-35 family during development, this family of miRNAs is an interesting model for studying the mechanisms of miRNA decay. The mir-35 family has two defining characteristics: the first is a shared, family-specific seed sequence (nucleotides 2-8 at the 5' of the miRNA), and the second is preferential loading into the Argonaute protein ALG-2 (rather than the better studied ALG-1). We are interested in ascertaining whether either of these characteristics play a role in the regulated decay of the mir-35 family. To examine if the mir-35 family turnover is seed sequence-specific, we mutated the seed sequence of the mir-35 miRNA via CRISPR and monitored levels of the mutant mir-35 as it compares to wild type mir-35. We detected perdurance of mutant mir-35 past embryogenesis, demonstrating the necessity of the seed sequence in regulating mir-35 family decay. We found that the mir-35 seed mutants were loaded into ALG-2 similarly to wild type mir-35, indicating that altered Argonaute loading is not the cause of the altered turnover of the mir-35 seed mutants. Therefore, ALG-2 loading is not sufficient for regulated turnover since mutant mir-35 is misregulated despite its normal loading into ALG-2. Having shown that the seed sequence is necessary for mir-35 decay, we next interrogated whether the seed is sufficient for the regulated decay of the mir-35 family. We mutated all residues of mir-35 outside of the seed region and observed that these miRNAs are decayed fairly similarly to wild type mir-35. Thus, the seed is largely sufficient to elicit developmentally timed decay. Overall, our findings contribute to a better understanding of the regulation of mir-35 family abundance in early development. By elucidating the mechanisms of mir-35 family decay, this research will offer insight into the broader mechanisms that couple miRNA decay to developmental progression.

Corpuz, Criszel et al. (2017) International Worm Meeting "HIS-35: a novel histone H2A protein involved in fertility."

Corpuz, Criszel, Jow, Margaret, Chu, Diana, Jimenez, Monet, Estrada, Rodrigo

[International Worm Meeting, 2017]

Infertility can arise when DNA accessibility is misregulated. This regulation is tightly controlled by core histones: H2A, H2B, H3, and H4 and the replacement with histone variants throughout germ line development. In C. elegans, three H2A variants were identified: HTZ-1, HTAS-1 and HIS-35. HTZ-1 is a known regulator of developmental genes while HTAS-1 is a unique component of sperm. However, the role of HIS-35, which differs from core H2A by only one amino acid is unknown. Preliminary data revealed HIS-35 enrichment in sperm and oocytes, thus I hypothesize that HIS-35 participates in germ line development to maximize fertility. To determine HIS-35 effects on fertility, we developed a mutant strain containing a deletion of the his-35 gene. Our results reported a 35% reduction in progeny, indicative of a role for HIS-35 in fertility. Furthermore, his-35; htas-1 double mutants are sterile, which is a phenotype more severe than either individual mutant alone. These results suggest that these H2A variants may compensate for one another to maximize fertility. Since we have demonstrated that HIS-35 is an important fertility factor, we utilized Crispr/Cas9 mediated repair system to create HIS-35::GFP and assess dynamics within the germ line. Surprisingly, HIS-35::GFP is present in almost all tissues and as expected, HIS-35::GFP localizes in both male and hermaphrodite germ lines. In addition, HIS-35::GFP is retained within mature sperm, oocytes and the newly-fertilized embryo. These findings raise the possibility that HIS-35 may be transferring or maintaining epigenetic states of genes needed throughout the developmental events of gametogenesis, fertilization and embryogenesis. Defining HIS-35 incorporation within these processes provides a basis for how H2A variants regulate DNA accessibility in different tissue types.

Liu M et al. (2011) Cell Res "mir-35 is involved in intestine cell G1/S transition and germ cell proliferation in C. elegans."

Fan Q, Zhang W, Liu D, Zhu Z, Cai Q, Zhang L, Gao G, Liu P, Liu M

[Cell Res, 2011]

MicroRNA (miRNA) regulates gene expression in many cellular events, yet functions of only a few miRNAs are known in C. elegans. We analyzed the function of mir-35-41 unique to the worm, and show here that mir-35 regulates the G1/S transition of intestinal cells and germ cell proliferation. Loss of mir-35 leads to a decrease of nuclei numbers in intestine and distal mitotic gonad, while re-introduction of mir-35 rescues the mutant phenotypes. Genetic analysis indicates that mir-35 may act through Rb/E2F and SCF pathways. Further bioinformatic and functional analyses demonstrate that mir-35 targets evolutionally conserved lin-23 and gld-1. Together, our study reveals a novel function of mir-35 family in cell division regulation.

Aaron M Bender et al. (2005) International Worm Meeting "Towards the cloning and characterization of the lin-35-synthetic gene, slr-10."

Aaron M Bender, David S Fay

[International Worm Meeting, 2005]

Using a previously described genetic screen (Fay et al, 2002) to identify genetic modifiers of lin-35/Rb, a mutant known as slr-10(fd6) has been isolated and partially characterized. The screen is conducted by mutagenizing lin-35(n745) mutant animals which carry an extrachromosomal array (kuEx119) containing wild type lin-35 as well as a ubiquitously expressed sur-5::GFP reporter. Upon loss of the array, animals harboring a mutation in a gene sharing genetic redundancy with lin-35 show synthetic phenotypes and are termed slr (Synthetic with lin-35/Rb). lin-35; slr-10 double mutants are small, thin, slow growing, and are markedly translucent compared to wild type or to either single mutant. Thus lin-35; slr-10 animals are superficially similar in appearance to previously characterized xnp-1, lin-35 mutants. In contrast to xnp-1, lin-35 mutants, however, lin-35; slr-10 double mutants are cross and self fertile but display a marked reduction in brood size compared to wild type strains. In addition, the self progeny of lin-35; slr-10 adults (derived directly the Ex+ parent) produce progeny that are either sterile or contain only inviable embryos. Furthermore, lin-35; slr-10 animals do not display the range and severity of gonad defects observed for xnp-1, lin-35 mutants. Nevertheless, the general gonad morphology of lin-35; slr-10 mutants appears to be abnormal and may result in improper formation and maintenance of a functional germline.slr-10 has been mapped to a small region on LGV using genetic and SNP markers. We have recently obtained full rescue of the lin-35; slr-10 mutant phenotype using a regional YAC and are current injecting available cosmids. In addition, we have used RNAi to identify candidate lin-35-interacting genes in the region in an effort to identify the slr-10 locus. Further progress towards identifying and characterizing slr-10 will be presented.

Julian Ceron et al. (2005) International Worm Meeting "Genome-wide RNAi identifies genes that genetically interact with lin-35 Rb"

Abha Chandra, Marc Vidal, John Satterlee, Sander Van Den Heuvel, Adriana K Jones, Jean-Francois Rual, Julian Ceron

[International Worm Meeting, 2005]

lin-35 is the sole C. elegans member of the retinoblastoma gene family. lin-35 Rb mutants display partial defects in fertility and growth, but are otherwise viable. Functions for lin-35 Rb have been uncovered in a variety of developmental processes by simultaneous inactivation of additional genes. Such studies revealed redundant functions for lin-35 Rb in vulval fate determination, cell-cycle control, pharyngeal morphogenesis, somatic gonad development, and meiosis. In order to uncover additional genes that act redundantly with lin-35 Rb we performed a genome-wide RNAi screen using ? 11000 ORFeome RNAi feeding clones to reduce gene functions in wild type and lin-35 Rb mutant animals. RNAi for 479 genes resulted in a synthetic or enhanced phenotype in the lin-35 Rb background as compared to wild type. This high number likely reflects the observed RNAi hypersensitivity of lin-35 and other class B synMuv mutants (Ruvkun lab, personal communication). Using several RNAi hypersensitive strains and double mutant combinations, we were able to identify a novel role for several genes interacting with lin-35 Rb in various developmental processes. For example, we have identified a zinc finger protein that cooperates with lin-35 Rb in somatic gonad formation. Furthermore, based on characterization of a deletion allele, we have identified a SWI/SNF complex subunit that cooperates with lin-35 in larval development. Interestingly, although we expected to find genes that act in parallel to lin-35 Rb, we found two novel synMuv B genes that could potentially act with lin-35 Rb in a multi-protein complex or pathway. The description of these mutant phenotypes as well as their genetic interaction with lin-35 Rb will be presented.

Fay, David S. et al. (2013) International Worm Meeting "Networks regulating pharyngeal development and morphogenesis."

Kuang, Jujiao, Karpel, Jon, Polley, Stanley, Yochem, John, Karina, Evguenia, Fay, David S., Kuzmanov, Aleksandra

[International Worm Meeting, 2013]

Development of the C. elegans pharynx is regulated by a network that includes the pRb ortholog, LIN-35, and PHA-1, a novel cytoplasmic protein. Deletion of pha-1 impairs pharyngeal development and body morphogenesis leading to a completely penetrant embryonic arrest. The lethality of pha-1 mutants is strongly suppressed by loss of function mutations in sup-35/ztf-21 and sup-37/ztf-12, which encode Zn-finger proteins, and by mutations in sup-36. Whereas sup-35 is non-essential, sup-37 is required in a subset of for pharyngeal muscle cells for pumping and also promotes ovulation in the somatic gonad. We have cloned sup-36 and shown it to encode a divergent Skp1 protein family member. Similar to SUP-35, SUP-36 protein levels are negatively regulated by a conserved E2-E3 complex composed of UBC-18/UBCH7 and ARI-1/HHARI. Unlike sup-35, however, sup-36 expression is not regulated by LIN-35-EFL-1 or HCF-1. Our analysis indicates that SUP-35/36/37 act at a single step to antagonize PHA-1 activity and inhibit pha-1 expression. Protein interaction studies show that SUP-35 and SUP-37 bind directly to each other and that SUP-35/37 may also be linked to SUP-36 via microtubule-associated cytoskeletal components. Specifically, SUP-35 binds to microtubules and SUP-36 interacts with the microtubule-associated protein PTL-1/tau. Consistent with these proteins acting within a complex, SUP-35 and SUP-36 display a similar dynamic pattern of expression during embryonic development. Because mutations in sup-35/36/37 also suppress the synthetic lethality of lin-35; ubc-18 double mutants, we sought to expand the pharyngeal regulatory network by identifying additional suppressors of lin-35; ubc-18. A genome-wide RNAi screen identified 39 suppressors, most of which are associated with transcriptional regulation. 23 of the genes that suppress lin-35; ubc-18 also suppress independent lin-35-synthetic phenotypes, consistent with these genes acting via the LIN-35 pathway. In addition, several Skp-1-Cullin-F-Box (SCF) components were identified, further indicating that SCF complexes are part of the greater network controlling pharyngeal development.

Michael Hurwitz et al. (2006) Development & Evolution Meeting "A Screen for Genes Synthetically Lethal with lin-35 Rb"

Bob Horvitz, Michael Hurwitz, Julia Den Uyl

[Development & Evolution Meeting, 2006]

The class B synMuv gene lin-35 encodes a C. elegans protein similar to Rb, a tumor suppressor inactivated in many human solid tumors. lin-35 mutants might provide a model for mammalian cells harboring mutant Rb genes. Because lin-35 null mutations are not lethal, we can screen for genes with functions required for viability in lin-35 mutants but not in wild-type animals to identify potential targets for cancer therapy. Pharmacologic inactivation of such target proteins could selectively kill Rb-deficient cells. We screened an LGI RNAi library1 for genes essential specifically in lin-35(n745) animals. n745 causes an early stop codon in lin-35 and is probably a null allele2. We have compared the phenotypes following RNAi of lin-35(n745) animals to the published RNAi phenotypes of the wild-type N2 strain. Of the 2445 dsRNAs we have screened, 2128 did not cause abnormalities in either lin-35(n745) or N2 worms. 212 dsRNAs caused similar abnormalities in lin-35(n745) and N2 worms. 105 dsRNAs resulted in abnormalities in lin-35(n745) but not in N2 worms. These 105 dsRNAs were tested in the RNAi-hypersensitive strain rrf-3(pk1426) to assess whether a more complete reduction of gene function by the dsRNAs generates more severe defects. Of the 105 dsRNAs,74 caused severe growth defects in rrf-3(pk1426) worms, suggesting that the effects seen in lin-35(n745) worms are related to the known increased sensitivity of lin-35 worms to RNAi3. To determine whether the effects of the 31 dsRNAs that caused apparent synthetic lethality in lin-35(n745) animals are strain- or allele-specific, we tested them on four other presumptive lin-35 null mutants (n2242, n2239, n2996, n373). In these strains, only three of the 31 dsRNAs caused lethality. Possibly, these other alleles are not nulls or the lin-35(n745) strain harbors background mutations that enhance the lethality of these dsRNAs. Currently, we are analyzing deletion alleles of lin-35 and the three genes that cause lethality in all of the lin-35 mutant strains to answer the above questions and to study the three likely positive genes identified in this screen. Also, we are screening the rest of the genome using the deletion allele of lin-35 that we have generated.