Kelley, Leanne et al. (2019) International Worm Meeting "Germline development and poly(U) polymerase activity."

Maine, Eleanor, Kelley, Leanne, McJunkin, Katherine, Li, Yini

[International Worm Meeting, 2019]

Poly(U) polymerases (PUPs) regulate RNA stability by addition of 3' uracil. 3' uridylation activity has been observed in many species, including Drosophila, S. pombe, humans, Arabidopsis, and C. elegans (Scott and Norbury 2013). We found that three enzymes with documented PUP activity, PUP-1/CDE-1, PUP-2, and PUP-3, are critical for germline development and survival (Li and Maine 2018). While single gene mutations reduce fertility, 100% of pup-1/-2 double mutants are sterile by the F3 generation at 25 deg C. In these animals, germ cells do not maintain their identity as distinct from soma and die during late larval development. Interestingly, germ cell viability is partially rescued in pup-3; pup-1/-2 triple mutants. Protein blots reveal that PUP-3 abundance is elevated in pup-1/-2 mutant gonads compared to wildtype controls. We propose that one way in which PUP-1 and PUP-2 promote germline development and viability is by limiting PUP-3 expression in the germ line. PUP-3 overexpression in the pup-1/-2 mutant germ line results in abnormal RNA regulation and improper germline development. We want to understand how the combination of PUP-1, -2, and -3 activity ensures fertility. Previous work showed that PUP-1 targets CSR-1-associated siRNAs (van Wolfswinkel et al. 2009) and PUP-2 targets let-7 miRNA in somatic tissues (Lehrbach et al. 2009). Human orthologs of PUP-1 and PUP-2 uridylate mRNAs (Lim et al. 2014), and we hypothesize that C. elegans PUPs may modify mRNAs, as well. To date, there has not been a complete analysis of PUP RNA targets in the C. elegans germline. We are identifying non-coding RNA targets using small RNA-seq and mRNA targets using TAIL-seq, a method that analyzes mRNA 3' ends. Preliminary data suggest that the relative distribution of small RNA classes is altered in pup-1/-2 double mutants compared to wildtype. MiRNAs as a class have reduced 3' uridylation in pup-1/-2 mutants. A subset of miRNAs, including members of the miR-35 family, have increased abundance, suggesting that PUP-1 and PUP-2 normally target these miRNAs for degradation.

Kelley, Leanne et al. (2021) International Worm Meeting "Characterizing the regulatory role of uridylation on small RNA activity in C. elegans germline development"

Maine, Eleanor, Ahmed-Braimah, Yasir, Li, Yini, Caldas, Ian, Sullenberger, Matthew, Kelley, Leanne, McJunkin, Katherine

[International Worm Meeting, 2021]

RNA regulation, from RNAi networks to maternally provided mRNAs, is essential for proper germline development and identity. Modifications to RNAs, such as the addition of nucleotides to the 3' end, can alter their function and affect biological activity. Poly(U) polymerases (PUPs), also known as terminal uridylyl transferases (TUTases) in vertebrates, place uridines on the 3' end of RNAs, an event called uridylation. While the presence of uridylation is well-documented on small non-coding RNAs (sRNAs) and mRNAs in many model organisms, the functional significance of uridylation is less clear. The uridylation catalyzed by PUPs usually mark the RNA for degradation, but other functions are being discovered. Identifying which sRNAs are being uridylated will provide a better understanding of how RNA tailing influences development. The roles of pup-1, pup-2, and pup-3 have previously been described in the C. elegans germline (Li and Maine 2018). Under temperature stress, the combined loss of pup-1 and pup-2 resulted in ectopic somatic expression in the germline, disorganized P-granules, and sterility by the third generation. In addition, pup-1 and pup-2 act redundantly to suppress pup-3 expression. In this work, we aim to connect the developmental defects of pup-1, pup-2, and pup-3 mutants to 3' uridylation activity on sRNAs. We performed sRNA-seq experiments to examine changes in sRNA expression and 3' tailing in pup-1, pup-2, and pup-3 deletion mutants. Uridylation is the most prevalent tail type, followed by adenylation, among siRNAs, miRNAs, and piRNAs. The overall proportion of uridylated sRNAs in pup-2(0) and pup-3(0) mutants are comparable to wildtype, while it is decreased in pup-1(0), pup-1/-2(0), and pup-3(0);pup-1/-2(0) mutants. When examining total sRNA abundance, pup-2(0) and pup-3(0) have few differentially expressed sRNAs compared to wildtype whereas pup-1(0), pup-1/-2(0), and pup-3(0);pup-1/-2(0) have many. Among ~2,670 differentially expressed sRNAs, 41% are shared between pup-1(0) and pup-1/2(0) while <1% are shared between pup-2(0) and pup-1/-2(0). However, 935 sRNAs are differentially expressed only in pup-1(0) and another 630 only in pup-1/-2(0), indicating that the combined loss of pup-1 and pup-2 has unique effect on sRNA expression. Future analysis will determine if uridylation is responsible for maintaining correct gene expression in the germline and how it affects different Argonaute-based classes of 22G-siRNAs.

Nikolaou S et al. (2019) Dev Cell "Invasion by Force: The C.elegans Anchor Cell Leads the Way."

Nikolaou S, Machesky LM

[Dev Cell, 2019]

How cells breach basement membrane barriers remains an area of active research. In this issue of Developmental Cell, Kelley etal. (2019) reveal that the C.elegans anchor cell uses physical force to breach basement membrane in the absence of matrix metalloproteases during its developmental invasion.

Carta LR et al. (1992) Worm Breeder's Gazette "Call for Worms"

Edgley ML, Thomas WK, Carta LR, Fitch DHA

[Worm Breeder's Gazette, 1992]

In response to the growing interest in worms related to C. elegans for comparative studies, we are coordinating an effort to collect a comprehensive array of cryopreservable species belonging to the order Rhabditida. This collection would be maintained at the Caenorhabditis Genetics Center, would be freely available to all interested scientists, and would provide an excellent resource for worm breeders who are interested in applying a broader phylogenetic viewpoint to comparative biological investigations. Of course, an important advantage of a universally accepted canonical set of living type species is that species identifications can be tested biologically through cross-mating experiments. The utility of such a collection has already been demonstrated in the Drosophila system a remarkable collection of species from around the world is maintained, for example, at the Bowling Green Stock Center in Ohio. We request that interested parties please send their wild isolates to David Fitch, Lynn Carta or Kelley Thomas, along with the following data: the date, source and method of isolation, any ecological information concerning the isolate, pertinent literature references, the names and addresses of the collector, the depositor and the taxonomist(1), and any specifics about stock maintenance. Other data about the species should also be included, such as measurements(2) and male tail characteristics. Scale illustrations and any anatomical, developmental, cytogenetic or molecular data are greatly appreciated. If the isolate is hermaphroditic, males should also be provided, since most of the morphological characters used in species identifications are associated with males. Males may occur spontaneously or can be induced by heat-shocking L4 or young adult hermaphrodites (usually, but not always, at 30 C for 6 hours). Males obtained in this way can be mated to hermaphrodites to maintain a stock containing males. Kelley will provide a molecular "identification tag". David and Lynn will determine if the species has been previously identified in the literature and serve as liaisons to nematologists with taxonomic expertise to help verify the species identification. We will then deposit the species with the CGC. Eventually, we hope to make all of the information associated with each species in the collection available in a database. (See the abstract by Fitch et al. in this issue for the latest information on current CGC species depositions). So hesitate not to share your pet species with us! We think that the effort in building a phylogenetically broad and comprehensive live collection of Rhabditida will be more than compensated by the valuable opportunities it will provide for developing novel approaches to many areas of nematode research.

Tram Do et al. (2007) International Worm Meeting "An RNAi screen for genes that function during spermatheca development."

Tram Do, Chris R. Gissendanner, Benji Morehead

[International Worm Meeting, 2007]

The spermatheca serves an important function in nematode reproduction. In C. elegans it is the site of sperm storage and fertilization of ovulated oocytes. The spermatheca is a highly secretory, tubular organ consisting of 24 epithelial cells. Passage of oocytes through the spermatheca is tightly regulated by specialized valve structures. Few genes have been identified that function during spermatheca development. However, our lab has recently determined that the nuclear receptor transcription factor NHR-6 is a critical regulator of spermatheca development (see abstract by Kelley et al.). NHR-6 is a member of the NR4A group of nuclear receptors that have important roles in mammalian organogenesis. The organogenesis functions of NHR-6 suggest that activities of this nuclear receptor group are conserved in C. elegans and that NR4A functions can be dissected using the C. elegans spermatheca as an organ model. To further understand the genetic control of spermatheca development we have initiated a genome-wide RNAi screen to identify spermatheca development genes. To specifically identify spermatheca genes we screen for a specific set of phenotypes known to be associated with nhr-6 mutants. The range of phenotypes include sterility, decreased brood size, abnormal egg morphology, and an adult degeneration phenotype that typically arises in nematodes with abnormally formed somatic gonad structures. Genes identified in this first phase of the screen are then re-screened using the spermathecal differentiation marker ajm-1::GFP. This screen also employed an RNAi sensitized background since nhr-6 is refractory to RNAi in the wild-type background, suggesting that other spermatheca genes may also be refractory to RNAi. We have completed a screen of chromosome III and have identified an initial set of 193 genes that are currently in the process of second round screening. Interestingly, many of the genes identified in our screen were not previously known to have reproductive functions.