Biology subjects

Kimble, J.

Publications and source records attributed to Kimble, J..

6 recordsLinked to original sources

bioRxiv2018

Niche maintenance of germline stem cells in C. elegans males

Stem cell maintenance by niche signaling is a common theme across phylogeny. In the Caenorhabditis elegans gonad, the broad outlines of germline stem cell (GSC) regulation are the same for both sexes: GLP-1/Notch signaling from the mesenchymal Distal Tip Cell (DTC) niche maintains GSCs in the distal gonad of both sexes (AO_SCPLOWUSTINC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWANDC_SCPLOW KO_SCPLOWIMBLEC_SCPLOW 1987), and does so via two key stem cell regulators, SYGL-1 and LST-1 (KO_SCPLOWERSHNERC_SCPLOW et al. 2014). Most analyses of niche signaling and GSC regulation have focused on XX hermaphrodites, an essentially female sex making sperm in larvae and oocytes in adults. Here we focus on XO males, which are sexually dimorphic in all tissues, including the distal gonad. The architecture of the male niche and the cellular behavior of GSCs are sex-specific. Despite these differences, males maintain a GSC pool similar to the hermaphrodite with respect to size and cell number and the male GSC response to niche signaling is also remarkably similar.

developmental biology↗

bioRxiv2018

Towards identifying subnetworks from FBF binding landscapes in Caenorhabditis spermatogenic or oogenic germlines

Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to \"disentangle\" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25{degrees}; for comparison, wild-type oogenic hermaphrodites were also raised at 25{degrees}. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.

developmental biology↗

bioRxiv2018

Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

molecular biology↗

bioRxiv2018

Nematode germ granule assembly is linked to mRNA repression

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P-granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P-granule scaffolding protein, called PGL, to investigate the functional relationship between P-granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL granules. We determine the crystal structure of the PGL N-terminal region to 1.5 [A], discover its dimerization and identify key residues at the dimer interface. In vivo mutations of those interface residues prevent P-granule assembly, de-repress PGL-tethered mRNA and reduce fertility. Therefore, PGL dimerization lies at the heart of both P-granule assembly and function. Finally, we identify the P-granule-associated Argonaute WAGO-1 as crucial for repression of PGL-tethered mRNA. We conclude that P-granule function requires both assembly and localized regulators.

developmental biology↗

bioRxiv2018

C. elegans germ cells divide and differentiate along a folded epithelium

Knowing how stem cells and their progeny are positioned within their tissues is essential for understanding their regulation. One paradigm for stem cell regulation is the C. elegans germline, which is maintained by a pool of germline stem cells in the distal gonad, in a region known as the progenitor zone. The C. elegans germline is widely used as a stem cell model, but the cellular architecture of the progenitor zone has been unclear. Here we characterize this architecture by creating virtual 3D models of the progenitor zone in both sexes. We show that the progenitor zone in adult hermaphrodites is essentially a folded epithelium. The progenitor zone in males is not folded. Analysis of germ cell division shows that daughter cells are born side-by-side along the surface of the epithelium. Analysis of a key regulator driving differentiation, GLD-1, shows that germ cells in hermaphrodites differentiate along the path of the folded epithelium, with previously described \"steps\" in GLD-1 expression corresponding to germline folds. Our study provides a three-dimensional view of how C. elegans germ cells progress from stem cell to overt differentiation, with critical implications for regulators driving this transition.

developmental biology↗

bioRxiv2016

An RNA binding polymer specifies nematode sperm fate

Metazoan germ cells develop as sperm or oocytes, depending on chromosomal sex, extrinsic signaling from somatic tissue and intrinsic factors within the germ cells. Gamete fate regulatory networks have been analyzed in nematodes, flies and mammals, but only in C. elegans have terminal intrinsic regulators been identified, which include a Tob/BTG protein family member, FOG-3. Canonical Tob/BTG proteins function as monomeric adaptor proteins that link RNA binding proteins to deadenylases. To ask if FOG-3 functions similarly, we first determined its crystal structure. FOG-3 harbors a classical Tob/BTG fold, but unlike other Tob/BTG proteins, FOG-3 dimerizes and these FOG-3 dimers assemble into polymers. The importance of FOG-3 polymers to sperm fate specification was confirmed in vivo using CRISPR/Cas9 gene editing to create mutations designed to disrupt the polymer interface. The FOG-3 surface potential is highly basic, suggesting binding to nucleic acid. We find that FOG-3 binds RNA directly with a strong preference for 3UTRs of oogenic mRNAs. Our results reveal a divergent but striking molecular assembly for proteins with a Tob/BTG fold, make key advances in understanding the mechanism of sperm fate specification and highlight the potential for undiscovered protein polymers in biology.

developmental biology↗