Finger, Fabian et al. (2015) International Worm Meeting "Regulation of protein homeostasis by microRNAs in Caenorhabditis elegans."

Finger, Fabian, Hoppe, Thorsten

[International Worm Meeting, 2015]

Maintenance of a functional set of proteins (proteome) is a highly dynamic process and crucial for any organism in terms of longevity and viability. A sophisticated machinery fulfills these demands in order to establish protein homeostasis (proteostasis) but its functionality declines with age. This machinery involves transcription, translation, protein folding, and degradation pathways, such as autophagy and the ubiquitin proteasome system (UPS). In this context, microRNAs, a class of short non-coding RNAs, might represent an elegant mechanism to control proteostasis. microRNAs are implicated in diverse biological processes including longevity; however, their influence on proteostasis mechanisms is not sufficiently investigated.Therefore, we started RNAi experiments inhibiting the biogenesis of microRNAs via knockdown of the essential factor PASH-1 and the argonaute proteins ALG-1 and ALG-2, respectively. Strikingly, downregulation of microRNA biogenesis results in a functional impairment of various proteostasis mechanisms, as indicated by reporter strains. Here, we especially made use of a ubiquitin fusion degradation (UFD) substrate, which is represented by a noncleavable Ubiquitin-GFP fusion protein. Accumulation of this substrate is easily detectable and strongly implicates malfunction of the UPS. In addition, ER-associated degradation (ERAD) and the unfolded protein response in the ER (UPRER) are also negatively affected upon downregulation of microRNA biogenesis. In order to identify microRNAs important for functional proteostasis, as well as facilitating downstream factors, we used a comparative approach that includes microRNA sequencing, microarray analysis, and microRNA target prediction algorithms. The comparison between unstressed and proteotoxic stress conditions allowed us to discover microRNAs involved in proteostasis mechanisms. Moreover, we are able to distinguish microRNAs with a broad influence from those affecting only specific pathways of the proteostasis machinery. Taken into account that regulatory processes can occur either cell-autonomously or cell-nonautonomously, we also tested tissue-specificity with particular attention to respective expression profiles.Our current observations suggest that we are able to identify microRNA families, as well as individual microRNAs that regulate diverse proteostasis mechanisms which might be the underlying cause of a functional impaired proteome influencing protein aggregation diseases and aging.

Finger, Fabian et al. (2021) International Worm Meeting "Olfaction regulates organismal proteostasis and longevity via microRNA-dependent signalling"

Hoppe, Thorsten, Ottens, Franziska, Finger, Fabian, Metz, Sophia, Ntogka, Andriana, Proksch, Lucie, Cochella, Luisa, Springhorn, Alexander, Drexel, Tanja

[International Worm Meeting, 2021]

The maintenance of proteostasis is crucial for any organism to survive and reproduce in an ever-changing environment, but its efficiency declines with age. Post-transcriptional regulators such as microRNAs (miRNAs) control protein translation of target mRNAs, with major consequences for development, physiology and longevity. Here we show that food odour stimulates organismal proteostasis and promotes longevity in Caenorhabditis elegans through miR-71-mediated inhibition of tir-1 mRNA stability in olfactory AWC neurons. Screening a collection of miRNAs that control ageing, we found that the miRNA miR-71 regulates lifespan and promotes ubiquitin-dependent protein turnover, particularly in the intestine. We show that miR-71 directly inhibits the Toll-receptor-domain protein TIR-1 in AWC olfactory neurons and that disruption of miR-71-tir-1 or loss of AWC olfactory neurons eliminates the influence of food source on proteostasis. miR-71-mediated regulation of TIR-1 controls chemotactic behaviour and is regulated by odour. Thus, odour perception influences cell-type-specific miRNA-target interaction, thereby regulating organismal proteostasis and longevity. We anticipate that the proposed mechanism of food perception will stimulate further research on neuroendocrine brain-to-gut communication and may open the possibility for therapeutic interventions to improve proteostasis and organismal health via the sense of smell, with potential implications for obesity, diabetes and ageing.

Y Hao et al. (2005) International Worm Meeting "Analysis of the RING finger protein PAR-2 implicates the ubiquitination machinery in polarity maintenance in the C. elegans zygote."

Y Hao, T Frazier, L Boyd, G Seydoux

[International Worm Meeting, 2005]

Polarization of the C. elegans zygote begins when a wave of actomyosin contractility sweeps the PAR-3/PAR-6/PKC-3 complex towards the anterior. During this time, the RING finger protein PAR-2 accumulates on the posterior cortex. PAR-2, although not essential to initiate polarity, is required to maintain polarity and prevent the return of PAR-3/PAR-6/PKC-3 (Munro et al., 2004; Cuenca et al., 2003).PAR-2 is an unusual protein with a RING finger domain and an ATP binding domain (Levitan et al., 1994; Boyd et al., 1996). We show that the RING finger, but not the ATP binding domain, is essential in vivo. We also identify a domain in PAR-2 necessary and sufficient for cortical localization. This domain contains several PKC phosphorylation sites. PKC phosphorylates PAR-2 in vitro, and mutations in the PKC sites cause GFP:PAR-2 to localize throughout the cortex in vivo. In contrast, mutations in the RING finger cause GFP:PAR-2 to become hypersensitive to PKC-3. PAR-2 can become ubiquitinated in vitro, suggesting that, like other RING finger proteins, PAR-2 may function as a ubiquitin ligase. We propose that phosphorylation of PAR-2 by PKC-3 prevents PAR-2 from localizing to the cortex and that the RING finger of PAR-2 allows it to overcome this inhibition in the posterior.

Joshua Snow et al. (2001) International C. elegans Meeting "Functional analysis of potential E2 ubiquitin conjugating enzymes using RNAi."

Rhonda Moore, Lynn Boyd, Claudia Morales, Joshua Snow

[International C. elegans Meeting, 2001]

We are investigating how genes predicted to be involved protein degradation effect embryogenesis in Caenorhabditis elegans . Within the cell, protein degradation is primarily accomplished through the ubiquitin-proteasome pathway. Studies in other systems show that E2 and E3 enzymes work in tandem to attach ubiquitin to a specific protein substrate, thereby condemning the substrate to degradation by the proteasome. We have identified 26 potential E2 genes within the completed genome of C. elegans . We are assessing the function of these genes through the use of RNAi-mediated interference (RNAi). E3 ligases are less conserved and more numerous than E2s. One class of E3 enzymes contains proteins with RING finger domains. We have previously identified 112 genes containing a RING finger in the C. elegans database. Four of the RING finger proteins were found to be required for embryogenesis (Moore, ECWM 2000, 154). By comparing E2 RNAi phenotypes with the RING finger mutant phenotypes, we hope to determine which E2 ubiquitin-conjugating enzymes partner with specific RING finger proteins. One of the four essential RING finger containing genes is par-2 , a gene involved in establishing anterior-posterior polarity in the embryo. PAR-2 protein is localized asymmetrically to the posterior cortex in embryos. In order to understand if protein degradation is involved in PAR-2 localization, we are using a transgenic strain expressing PAR-2:GFP to observe PAR-2 localization in E2 RNAi embryos.

Scott W Knight et al. (2005) International Worm Meeting "Characterization of a Zn-finger protein involved in RNAi"

Scott W Knight, Anhtram T Nguyen, Xavier W Belcher

[International Worm Meeting, 2005]

One of the earliest steps in the RNAi mechanism is the cleavage of the initiating double-stranded RNA into small interfering RNAs (siRNAs) by the RNaseIII-like enzyme Dicer. Animals with mutations in the gene encoding Dicer (dcr-1) are RNAi defective, exhibit heterochronic phenotypes, and are sterile. To further understand the initial step in the RNAi pathway, we developed a screen to identify genes encoding proteins that may function in siRNA production or stability. Our screen was designed to identify animals that were RNAi defective and had developmental defects similar to those observed in dcr-1/dcr-1 animals. We identified a number of genes that appear to be required for an effective RNAi response, including a gene encoding a protein with multiple Zn-finger domains. Although often thought of as DNA-binding proteins, a number of Zn-finger proteins bind RNA. Animals containing a deletion in the gene encoding the Zn-finger protein grow slowly and have defects in germ line development that lead to sterility. We are currently investigating the role of this gene in siRNA and miRNA biogenesis, and ongoing experiments are designed to characterize the role of the Zn-finger protein in germ line development.

Batchelder C et al. (1997) International C. elegans Meeting "TRANSCRIPTIONAL REPRESSION BY THE PIE-1 PROTEIN"

Mello CC, Batchelder C, Choy B, Cassie C, Suh Y, Blackwell TK

[International C. elegans Meeting, 1997]

PIE-1 is a maternally-expressed protein which is required for germline specification in the early C. elegans embryo. PIE-1 appears to maintain the germ cell precursors in a transcriptionally inactive state, which may be a mechanism to ensure that their fate is not restricted and remains pluripotent. We are interested in establishing if PIE-1 acts directly as a transcriptional repressor, and in elucidating how it functions. PIE-1 is composed of 335 amino acids and contains 2 putative zinc fingers (C3H) in its central region. In order to examine the transcriptional activity of PIE-1, we have set up a transient transfection assay system in mammalian cells. We have observed that PIE-1 can repress transcription from many promoters transcribed by RNA polymerase II, and inhibit a variety of transcriptional activators, including SKN-1. To determine which sequences have repression activity, we have divided PIE-1 into 3 regions (N-terminal, central zinc finger and C-terminal) which were fused to the GAL4 DNA-binding domain. Both the zinc finger and C-terminal regions repressed transcription, the C-terminal region being the stronger repressor by several fold. The C-terminal region was dissected into several sub-regions in order to more precisely define the sequences that mediate the repression. A smaller region with repression activity has been identified and we are currently evaluating the significance of sequence motifs within the region, and whether they might be involved in an interaction with the transcriptional machinery. To further study the function of the PIE-1 zinc finger region, we are presently making several zinc finger mutations, and are also comparing the transcriptional activity of PIE-1 with that of other C3H zinc finger proteins.

Cynthia L DeRenzo et al. (2005) International Worm Meeting "Exclusion of germ plasm components from somatic lineages by localized protein degradation."

Kimberly J Reese, Cynthia L DeRenzo, Geraldine Seydoux

[International Worm Meeting, 2005]

In Drosophila, C. elegans, Xenopus and Zebrafish embryos, germ plasm components segregate with the germ lineage and are not maintained in somatic lineages. This segregation of the germ plasm is generally thought to involve active targeting of germ plasm components to a specific region of the oocyte and/or embryo. However, we have shown that germ plasm asymmetry also depends on a mechanism that targets germ plasm proteins for degradation in somatic cells. We have identified a novel protein ZIF-1(Zinc-finger Interacting Factor-1) that is required for the somatic degradation of five C.elegans germ plasm proteins. These five proteins all share a common CCCH zinc-finger motif that is required both for somatic degradation as well as for binding to ZIF-1. ZIF-1, a BC-box protein, also binds to the E3 ubiquitin ligase subunit elongin C. Elongin C, the cullin CUL-2, the ring finger protein RBX-1 and the E2 ubiquitin conjugation enzyme UBC5 are all required in vivo for ZIF-1-dependent degradation. These observations are consistent with ZIF-1 functioning as a substrate-recruitment factor of an E3 ubiquitin ligase that targets CCCH finger proteins for degradation in somatic cells. Interestingly, we have found that two of these CCCH finger proteins, MEX-5 and MEX-6 are required to activate degradation. In mex-5(-);mex-6(-) mutants, ZIF-1-dependent degradation does not occur. In contrast, in par-1(-) mutants where MEX-5 and MEX-6 levels are high in all cells, degradation occurs in all cells. MEX5/6 have previously been shown to regulate many anterior-posterior differences in the embryo (Schubert et al. 2000) and therefore, may activate ZIF-1 indirectly. However, like other CCCH finger proteins MEX-5/6 bind to ZIF-1 and are themselves targets of ZIF-1-dependent degradation. We are currently testing whether the binding of MEX5/6 to ZIF-1 is required to activate degradation. REFERENCES: Schubert, C.M. et al. (2000) MEX-5 and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos. Mol. Cell 5, 671682.

Anastasiades, Daphne et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Investigation of the molecular function of the chromatin factor Zinc Finger Protein 1, ZFP-1, in C. elegans"

Anastasiades, Daphne, Grishok, Alla, Cecere, Germano, Mansisidor, Andres

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Transcriptional gene silencing directed by small interfering RNAs is intimately linked to the formation of heterochromatin in S. pombe. Our research focuses on studying the molecular mechanisms and biological functions of the RNAi processes that regulate chromatin organization and transcription in C. elegans. Zinc Finger Protein 1 (ZFP-1), the C. elegans PHD finger-containing protein homologous to the human chromatin-associated protein AF10, is one of the factors affecting RNAi- transcriptional gene silencing (TGS). Our previous data suggest that ZFP-1 may be central for the regulation of endogenous genes by a large class of short interfering RNAs (siRNAs) antisense to their targets (endo-siRNAs). ZFP-1/AF10 has two N terminal PHD fingers. Here, using in vitro binding assays, we report that the first PHD finger of ZFP-1 binds specifically to the dimethylated state of lysine 4 on histone H3 (H3K4me2). Our preliminary data also suggests that the second PHD finger may be involved in dimerization of the protein. Using a ZFP-1::GFP translational fusion reporter, we observe nuclear localization of ZFP-1 in most cell types as well as ZFP-1 localization to the chromosomes in oocytes. Specifically, staining in oocytes with anti-H3K4me2 antibodies shows co-localization of ZFP-1 with the H3K4me2 mark. Furthermore, because methylation of H3K4 accompanies transcription, we propose that ZFP-1 may connect RNAi factors with transcriptional machinery to regulate target gene expression.

T Shin et al. (1999) International C. elegans Meeting "Zinc-finger and KH domain Proteins in C. elegans Embryogenesis"

CC Mello, T Shin

[International C. elegans Meeting, 1999]

In the early embryo, the expression of maternally encoded proteins is controlled both spatially and temporally. Two groups of maternal proteins have been implicated in this process. The first consists of Cys3-His zinc-finger proteins including PIE-1, MEX-1, MEX-5/AH6.5 and POS-1. The second class consists of KH domain proteins including ALP-1, MEX-3 and GLD-1. Mutations in most of these genes cause embryonic cell fate transformation, and in some cases, are correlated with clear misexpression of maternal proteins. Both Cys3-His zinc-fingers and KH domains are conserved in a wide variety of organisms and in some cases are implicated in direct RNA binding, raising the possibility that these two families of proteins directly regulate maternal mRNA translation. We identified ALP-1 as a two-hybrid interacter of PIE-1. This interaction requires a part of ALP-1 KH domain and a C-terminal region in PIE-1 outside of its zinc-fingers. We also found that while ALP-1 binds only to PIE-1 among all zinc-finger proteins tested, other KH domain and zinc-finger proteins can interact with multiple partners. For example, GLD-1 can bind strongly to POS-1 and weakly to PIE-1, and POS-1 can interact with MEX-3. These data suggest that zinc-finger/KH domain proteins may function together as a complex and/or regulate each other's activity. Consistent with this view, gld-1(RNAi) produces a no-gut defect very similar to that caused by pos-1 mutations. Furthermore, alp-1 interacts genetically with pos-1 and gld-1 , completely suppressing the no-gut phenotype in both. Thus, the two protein families appear to regulate each other in a complex manner. How do zinc-finger/KH domain proteins function in controlling maternal mRNA translation? Do they directly recognize RNA? What are the biochemical bases for their apparently complex interactions both physically and genetically? Finding answers for these questions will require in vivo biochemical analyses. Toward this end, we have generated transgenic animals that stably express functional HA-tagged PIE-1 protein. We are currently examining in vivo protein-protein interaction between HA-PIE-1 and other members of the two protein families. These studies should shed light on biochemical and physiological roles for the evolutionarily conserved zinc-finger/KH motif proteins.

Anastasiades, Daphne et al. (2009) International Worm Meeting "Investigation of the molecular function of the chromatin factor Zinc Finger Protein-1, ZFP-1, in C. elegans."

Cecere, Germano, Anastasiades, Daphne, Grishok, Alla, Mansisidor, Andres

[International Worm Meeting, 2009]

The discovery of RNA interference (RNAi) has led to a new understanding of the regulation of gene expression. Currently, both nuclear and cytoplasmic RNAi have been observed in C. elegans. Zinc Finger Protein 1 (ZFP-1), a PHD finger containing protein that is the C. elegans homolog of the human chromatin-associated protein AF10, is one of the genes affecting nuclear RNAi. Our additional data suggest that ZFP-1 may be central for the regulation of endogenous genes by a large class of short interfering RNAs (siRNAs) antisense to their targets (endo-siRNAs). The molecular function of ZFP-1 is not known and is thus the primary focus of this investigation. We find via histone binding assays in vitro that the first PHD finger of ZFP-1 binds specifically to the dimethylated state of lysine 4 on histone H3 (H3K4me2). Using a ZFP-1::GFP translational fusion reporter, we observe nuclear localization of ZFP-1 in most cell types as well as ZFP-1 localization on the chromosomes in oocytes. Staining in oocytes with anti-H3K4me2 antibodies shows co-localization of ZFP-1 with the H3K4me2 mark, which correlates with our in vitro findings. Furthermore, because methylation of H3K4 accompanies transcription, we propose that ZFP-1 may connect RNAi factors with transcriptional machinery.