Carrick, Brian et al. (2017) International Worm Meeting "Genomic analyses of interactions that govern an RNA regulatory network in stem cells."

Guthrie, Timothy, Wickens, Marvin, Carrick, Brian, Kimble, Judith, Kroll-Conner, Peggy, Prasad, Aman, Porter, Douglas

[International Worm Meeting, 2017]

mRNA regulation governs when, where, and how much protein an mRNA produces and thereby determines cell identity, function and behavior. A single mRNA regulatory protein can bind to and control hundreds to thousands of mRNAs, forming a "network" that enables control of a biological outcome. This work focuses on C. elegans FBF-1 and FBF-2 (collectively termed FBF), which maintain germline stem cells (GSCs). FBF is a member of the PUF (Pumilio and FBF) family of RNA-binding proteins (RBPs), which bind mRNAs via sequence-specific elements in the 3' untranslated region (3'UTR) and regulate expression. We have now identified the RNAs bound by FBF in vivo in both oogenic and spermatogenic germlines using the technique of iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPreciptation). The goal of this work is to learn FBF targets that function independently of gender, as well as gamete-specific targets and to thereby gain insight into FBF sub-networks. Like other PUF proteins, FBF acts with protein partners which can modulate binding specificity in vitro. The challenge now is to understand how these partners affect FBF regulatory networks in vivo. Currently, we are delineating protein - protein interfaces of FBF with its partners and generating CRISPR-induced mutants that abrogate those partnerships. Ultimately we will analyze how the FBF subnetworks fluctuate in response to changing FBF partnerships.

G Aspoeck et al. (1999) International C. elegans Meeting "A second look at warthog and groundhog genes"

G Aspoeck, H Kagoshima, TR Buerglin

[International C. elegans Meeting, 1999]

The hedgehog family of signaling glycoproteins have many essential functions in animal development. They are secreted by "posterior" cells in fly parasegments, wing discs, leg discs and vertebrate limb buds and also define subgroups of neurons in the central nervous systems of flies and vertebrates. Hedgehog proteins consist of two domains: the carboxyterminal autoprotease/ cholesterol transferase cleaves off the secreted amino-terminal signal and anchors it to a cholesterol moiety, thus to the membrane. C. elegans apparently does not have a hedgehog homolog (although there is a slim chance that it has not been sequenced yet). However, several C. elegans genes contain the carboxy-terminal autoprotease domain. We call it hog domain; it is also referred to as Hint domain because of the similarity to inteins, bacterial self-splicing proteins (Porter et al.,1996). The hog domains are associated with novel amino-terminal sequences that are up to now unique to nematodes. Using wart and ground as names for these domains C. elegans has five warthog and three goundhog genes. One gene has an orphan amino-terminus (quahog), and there is a presumed pseudo-gene, consisting only of a hog domain. Additionally there are 5 wart-only and 12 ground-only genes, 2 ground-derived genes, and 28 ground-like genes which all lack a hog domain. wart, warthog, ground, and ground-like homologues are also present in parasitic nematodes. Here we present sequence comparisons, gfp expression patterns and some RNAi experiments of wrt and grd genes. Several wrt gfp reporter constructs are expressed in the hypodermal syncytia; expressions of other wrt genes include seam cells, socket and sheath cells of anterior and phasmid sensilla, gonad sheath cells, spermatheca, uterus, the pharyngeal gland cell g1, the excretory cell, pharyngeo-intestinal valve cells and rectal cells. Only three grd reporter constructs are expressed; we find gfp in rectal cells, motor and other neurons. References: Porter et al. (1996). Cell 86:21-34. Burglin (1996) Curr. Biol. 6:1047-1050

Wang X et al. (1998) East Coast Worm Meeting "A MOLECULE CONTAINING A HEDGEHOG-LIKE AUTOPROCESSING DOMAIN IS REQUIRED FOR MOLTING IN C. ELEGANS."

Beachy P, Seydoux GC, Wang X

[East Coast Worm Meeting, 1998]

The Hedgehog family of signaling molecules controls many aspects of animal development, from embryonic patterning to limb formation. Recently, these proteins have been shown to rely on a novel mechanism to regulate their spatial distribution (1,2). Hedgehog proteins undergo an autoprocessing reaction to yield two distinct products, an amino-terminal fragment (Hh-N), and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms may be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a set of eight C. elegans proteins with sequence similarity to the Hh-C domain of Hedgehog (2,3). In these proteins, as in the Hedgehog family, the Hh-C-like domain is located in the carboxy-teminal end, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). This structure is consistent with the possibility that these proteins are secreted and undergo processing in a manner similar to that described for Hedgehog. To test this possibility we have fused the Hh-C-like domain of one of these proteins to a His tag, and purified the fusion protein from E. coli. We find that such a fusion can undergo the processing reaction in vitro, suggesting that the Hh-C-like domain is functionally active in the nematode proteins. We have begun to characterize the functions of Hh-C-related proteins using RNA-mediated interference. Our initial results suggest that one of these proteins, T05C12, is essential for molting. Injection of T05C12 double-stranded RNA results in 90% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. In many animals, a cuticular plug can be seen obstructing the mouth opening where it presumably interferes with feeding. The amino-terminal domain of T05C12 contains several sequence motifs similar to those found in collagens and other extracellular matrix proteins, raising the possibility that the T05C12 product is a cuticle component. Consistent with this possibility, a T05C12:GFP fusion is expressed in hypodermal cells during larval and adult stages. We are currently determining the effects of expressing mutant forms of T05C12 to test the function of its Hh-C-like domain. 1. Porter, JA. et al. (1996). Cell 86, 21-34. 2. Porter, JA. et al. (1996). Science 274, 255-259. 3. Burglin, TR. (1996). Current Biology 6, 1047-1050.

Crittenden, Sarah et al. (2021) International Worm Meeting "FBF binding elements in the gld-1 3'UTR and their role in germline regulation"

Woodworth, Jennifer, Kimble, Judith, Kroll-Conner, Peggy, Wickens, Marv, Crittenden, Sarah

[International Worm Meeting, 2021]

PUF RNA binding proteins (for Pumilio and FBF) are key regulators of self-renewal in the C. elegans germline. Two nearly identical PUF proteins, FBF-1 and FBF-2 (collectively FBF), have long been known as self-renewal regulators of late larval and adult germline stem cells (GSCs) (1). Genomic studies using iCLIP identified a battery of FBF target RNAs as well as FBF binding elements (FBEs) within them (2). That knowledge of in vivo FBEs provides a powerful inroad for studying the direct effects of FBF binding on regulation of individual target RNAs and germ cell fate. A major target of FBF regulation is gld-1, which promotes meiotic progression and oogenesis (3). Strong evidence has accumulated for FBF repression of gld-1 in GSCs (1), but FBF had also been proposed to activate gld-1 to promote meiotic entry. (1,4). We have now begun to focus on FBEs in the gld-1 3'UTR to assess their molecular and biological roles. To this end, we used CRISPR/Cas9 gene editing to mutate the two canonical gld-1 FBEs, FBEa and FBEb, in the 3'UTR of endogenous gld-1. Phenotypically, FBEa and FBEb single mutants both maintain GSCs and are fertile; an FBEa FBEb double mutant was just recovered and is being characterized. Using GLD-1 immunostaining, we find that the FBEa single mutant makes higher than normal levels of GLD-1 protein in GSCs, while the FBEb single mutant makes wild-type levels in GSCs. We are currently examining gld-1 mRNA levels in these single mutants using smFISH, and soon will start analyzing the double mutant. 1 Crittenden et al.(2002).Nature, 417,660. 2 Porter, Prasad et al.(2019).G3,9,153. 3 Francis et al (1995) Genetics 139, 579 4 Suh et al.(2009).Genetics,181,1249.

Aspock G et al. (2000) European Worm Meeting "The warthog gene wrt-6 is required for proper formation of amphids and phasmids"

Burglin TR, Kagoshima H, Aspock G

[European Worm Meeting, 2000]

Of the classical hedgehog signalling pathway C. elegansposesses only fragments. Especially the hedgehog signal itself has not been found in the genome, although a few genes resemble the hedgehog receptor patched. Examples not only from C. elegans show however that signalling pathways do not always require the same complete set of molecules. Ten genes in the worm genome have a carboxyterminal autocleavage domain that is typical of hedgehog family genes. This HINT/hog domain cleaves off the aminoterminal active signalling part and anchors it to a cholesterol moiety (Porter et al., 1996). The N-terminal 'signals' of these C. elegansgenes - if they are signals - are not related to the hedgehog N-terminus (except for a four amino acid motif of unknown function that hints at a common evolutionary relationship between hedgehog and the C. elegans genes). We therefore termed groups of genes with similar amino termini warthog and groundhog in analogy to hedgehog. These genes are expressed in overlapping patterns mainly in nonneural ectodermal tissues (Aspock et al., 1999). We are interested to find out whether these genes really constitute signals, and what their message is. We have isolated a deletion mutant in wrt-6, a gene expressed in a few sheath and/or socket cells of anterior sensilla. The mutant is 100% dye-filling defective in amphids and phasmids; occasionally a few amphid neurons fill but never all. We assume that the defect is structural and will perform EM sections to characterize it. To find out whether the gene acts cell-nonautonomously and thus may be a signal we are attempting to rescue the wrt-6 mutation by expressing wrt-6 cDNA from ectopic promoters. Interestingly, the wrt-6 mutant dye-filling pattern similar to that of che-14, a gene related to the hedgehog receptor patched (see abstract by Michaux et al.). A comparison of EM sections will show whether their phenotypes are related also at a structural level.

Wang, X. et al. (2019) International Worm Meeting "The balance of activities of PUF family proteins FBF-1 and FBF-2 coordinately regulates germline stem cell proliferation and differentiation in C. elegans."

Voronina, E., Hickey, B., Wang, X., Ellenbecker, M., Day, N., Baumgarten, E.

[International Worm Meeting, 2019]

Studies of the molecular mechanisms regulating the balance of stem cell proliferation and differentiation can help us understand the causative factors for ageing, cancer and various degenerative disorders. Pumilio and FBF (PUF) family RNA-binding proteins are highly conserved regulators of stem cell development. Here, we report that FBF-1 and FBF-2, two PUF proteins in C. elegans, coordinately change the rate of both proliferation and differentiation of germline stem cells. FBF-1 and FBF-2 function redundantly in maintaining germline stem cells through translational repression of target mRNAs (Crittenden et al., 2002). FBF-1 and FBF-2 are very similar in primary sequence and share most of their target mRNAs (Prasad et al., 2016, Kershner et al., 2010 and Porter et al., 2018), but recently we found that they have distinct effects on proliferation and differentiation of stem cells. The analysis cell cycle parameters indicated that FBF-1 might slow down cell proliferation while FBF-2 accelerates cell proliferation of germline stem cells. Additionally, estimating the rate of meiotic entry suggested that FBF-1 slows down differentiation in concert with slowing down proliferation. Quantification of FBF target mRNA abundance by qPCR suggested that the presence of FBF-1 correlates with degradation of target mRNAs regulating proliferation and differentiation, while FBF-2 protects these targets from degradation. We propose that the balance of FBF-1 and FBF-2 activities might determine the steady-state abundance of target mRNAs and thus change the rate of both cell cycle and meiotic entry. Furthermore, we found that FBF-1 activity in stem cells requires CCR4-NOT deadenylase machinery that shortens poly(A) tail of mRNA. By contrast, FBF-2 activity in stem cells shows less dependence on CCR4-NOT, which correlated with FBF-2's interaction with its specific cofactor, DLC-1. In conclusion, our study suggested a new mechanism for control of stem cell proliferation in concert with differentiation by PUF proteins through differential cooperation with CCR4-NOT deadenylase machinery.

X Wang et al. (1999) International C. elegans Meeting "A molecule containing a Hedgehog-intein (HINT) domain is required for molting in C. elegans."

G Seydoux, P Beachy, X Wang

[International C. elegans Meeting, 1999]

Hedgehog proteins undergo an autoprocessing reaction to yield an amino-terminal fragment (Hh-N) and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C, which consists of a Hint domain and a sterol recognition region, mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms might be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a C. elegans protein (T05C12.10) with sequence similarity to the Hint domain of Hedgehog (Porter, JA. et al.,1996a,b; Burglin, TR., 1996). In this protein, as in the Hedgehog family, the Hint domain is located near the carboxy-terminus, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). We have shown that the Hint domain of T05C12.10 can lead to the formation of an internal thioester intermediate in vitro . We have begun to characterize the functions of the T05C12.10 using RNA-mediated interference. Our results suggest that T05C12.10 is essential for molting. Injection of T05C12.10 double-stranded RNA results in 100% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. This phenotype resembles the phenotype of worms starved for cholesterol and of mutants lacking the megalin homologue, lrp-1 (Yochem, J. et al. 1999). The amino-terminal domain of T05C12.10 contains several sequence motifs similar to certain extracellular matrix proteins. Consistent with this, T05C12.10 protein colocalizes with megalin in the apical surface of hypodermal cells during larval and adult stages. Western analysis indicates that there are two forms of T05C12.10 in vivo , and that the relative abundance of the smaller form increases during each molt. These observations suggest that processing of T05C12.10 coincides with, and may be regulated by, molting. We are currently determining the identity of the smaller form as well as testing its role in molting.