Jean-Claude Labbe

University of Montreal; Montreal QC, Canada; Institute of Research in Immunology and Cancer (IRIC) University of Montreal; Montreal QC, Canada; Department of Pathology and Cell Biology

Labbe J-C et al. (1998) East Coast Worm Meeting "Ongoing characterization of rop-1: an update"

Rokeach LA, Labbe J-C, Hekimi S, Plasterk RHA

[East Coast Worm Meeting, 1998]

Ro ribonucleoproteins have been initially identified as immunological targets in patients suffering autoimmune diseases such as systemic lupus erythematosus and Sjogren's syndrome. In most human cells, these particles are composed of at least one protein of 60 kDa, Ro60, bound to 1 of 4 small cytoplasmic RNAs, designated Y RNAs. Although Ro60 has been proposed to participate in the quality control of 5S rRNA in Xenopus oocytes, the function of the ribonucleoprotein remains elusive. In order to investigate in vivo the function of Ro60, we have previously cloned the gene rop-1, encoding the homologue of Ro60 in C. elegans. We obtained a strain carrying the allele pk93 in which rop-1 is disrupted by the insertion of a Tc1 transposon element. This insertion results in a null allele since no mRNA was detected by RT-PCR and no ROP-1 could be detected in total protein extracts, using a ROP-1 specific antibody. Interestingly, no visible phenotype was associated with this disruption, even when worm phenotypes such as brood size and life span were scored. This result is intriguing considering the level of conservation of ROP-1 throughout species. Nevertheless, at the molecular level, we observed a dramatic decrease in the levels of another component of the Ro ribonucleoproteins, the Y RNA (CeY RNA). We were able to partially rescue CeY RNA levels by re-expressing wild type rop-1 in the rop-1 mutant, thereby demonstrating the specific link between this molecular phenotype and rop-1. Taken together, our results demonstrate that ROP-1 does not play a crucial role in C. elegans, but might be regulating the levels of CeY RNA.

Labbe J-C et al. (2000) East Coast Worm Meeting "nop-1 and the establishment of asymmetries in the early C. elegans embryo"

Labbe J-C, Goldstein B

[East Coast Worm Meeting, 2000]

The generation of asymmetries before the first cell division in the early C. elegans embryo appears to be a complex mechanism that involves the activity of several genes. Cortical and cytoplasmic flows are among some of the first observed aspects of asymmetry. These flows appear to play important role(s) in the generation of early asymmetries, as many cellular components become localized concomitantly with them, and disruption of the actin cytoskeleton by treatment with cytochalasin D at the time of flows also affects embryonic asymmetries (Hill and Strome, 1990). We are interested in understanding how asymmetries are established in the early C. elegans embryo. One of the genes that might participate in some of these early events is nop-1. nop-1(it142) was shown by Rose et al. (1995) to be a viable mutant that lacks a pseudocleavage furrow, and has reduced flows. Furthermore, 20% of embryos fail to hatch. We were interested to study in more detail the phenotype of nop-1(it142), in order to find out whether the embryos that dont hatch also have asymmetry problems. We found that a proportion of the nop-1 mutant embryos had no cytoplasmic flows; these embryos failed to hatch. Interestingly, most of these defective embryos either had a Par phenotype or failed to complete first cytokinesis. Some of the nop-1 mutant embryos that did have flows also failed to hatch, although their lethality did not appear to result from a defect in early asymmetries, but might be explained by the observation that nop-1 mutant animals have a weak Him phenotype, suggesting a defect in chromosomal segregation. We are currently examining the possibility that nop-1 might genetically interact with other genes involved in early embryogenesis. We set out to clone nop-1 as a step toward further defining its role in early embryogenesis. This will help us to address the null phenotype of nop-1, as it142 might be a weak loss-of-function allele. nop-1(it142) was previously mapped by Lesilee Rose between dpy-17 and unc-32, at approximately 1.5 cM on the left arm of chromosome III. We refined the mapping using several deficiencies in that area and obtained partial rescue with one YAC in this region. We are currently assessing rescue with cosmids in the region and will present further details at the meeting.

Picture from Labbe J-C et al. (1999) Genetics "The levels of the RoRNP-associated Y RNA are dependent upon the presence of ROP-1, ...."

Figure 5. Transgenic expression pattern of rop-1. X-Gal staining of whole animals transformed with the in-frame rop- 1::lacZ reporter construct pCeRo4107. Eight independent strains were assayed for b-galactosidase staining, and the expression patterns from individual worms were compared. The transgenic rop-1 promoter is expressed in every cell type of the organism, except in the germline. This photograph shows representative results obtained by this method.

Picture from Labbe J-C et al. (1999) Genetics "The levels of the RoRNP-associated Y RNA are dependent upon the presence of ROP-1, ...."

Figure 4. CeY RNA is transcribed at higher levels in the adult germline and in embryos. Total RNA was prepared from staged worms and 5 ug of total RNA was resolved by formalde- hyde-agarose gel electrophoresis, transferred, and probed with (A) a rop-1 cDNA, (B) the yrn-1 gene, or (C) a portion of the gene encoding actin. The levels of rop-1 mRNA and CeY RNA are both high in the embryos, but decrease at the subsequent larval stages. However, the levels of CeY RNA increase a little at the L4 stage and a lot at the adult stage. Lane 1, wild-type embryos; lane 2, wild-type L1 larvae; lane 3, wild-type L2 larvae; lane 4, wild-type L3 larvae; lane 5, wild-type L4 larvae; lane 6, wild-type nongravid adults; lane 7, glp-1(q231) mutant adults. (D) The intensity of each band obtained in (B) was deter- mined by scanning the X-ray film with a LKB ultroscan XL enhanced laser densitometer (Pharmacia, Piscataway, NJ). Each individual band in (B) was corrected with the densitomet- ric value obtained by scanning the band in the corresponding lane obtained in (C) with an actin probe. The corrected data were plotted on a graphical chart.

Labbe J-C et al. (1996) East Coast Worm Meeting "Molecular and genetic studies of the Ro ribonucleprotein genes in C. elegans"

Labbe J-C, Rokeach LA, Hekimi S

[East Coast Worm Meeting, 1996]

The sera of patients suffering from autoimmune diseases such as systemic lupus erythematosus and Silgren's syndrome often contain autoantibodies that target components of the Ro ribonucleoproteins (RoRNP). In human, these RoRNP are madeup of at least one major protein of 60 kDa, Ro60, and one of four small RNAs, hY RNAs. The La protein, which is a RNA polymerase III transcription termination factor, is also transiently associated with the complex by binding the poly(U) tail of the hY RNA. Although Ro60 has been implicated in the quality control of 5S rRNA production, the cellular role(s) of RoRNP remains elusive. In order to establish a genetic system for the the study of RoRNP genes, we have isolated the gene rop-1, encoding the C. elegans homologue of Ro60. A single copy of this gene is located on the right arm of chromosome V, near genetic marker unc-42 (cosmid C47A8), and it contains a 643 codon long open reading frame which is interupted by three introns. The encoded protein, Rop1p, shares 40% identity and 60% similarity with both human and Xenopus Ro60. Expression studies in transgenic N2, using a construct that places lacZ under the control of rop-1 promoter, indicate that rop-1 is expressed in every somatic cell of the organism. Disruption of rop-1 by insertion of transposon Tc1 results in the production of truncated mRNA, leading to a complete knock-out of the expression of Rop1p. Moreover, we observe an increase in the level of the aberrant mRNA compared to wild type, which could suggest an autoregulation of the rop-1 promoter by Rop1p itself. Nevertheless, no visible phenotype is associated with this deletion. This result is quite surprising considering the level of conservation of Rop1p throughout species. We have recently sequenced a cDNA, yk74a1, encoding the C. elegans homologue of the La protein (CeLa). This protein contains 445 amino acids (50 kDa) and is fairly similar to its homologues in other species. The gene encoding CeLa is located on chromosome I, near genetic marker unc-57 (cosmid C44E4). Interestingly, immunoprecipitation of Rop1p using specific anti-Rop1p antibodies show a stable association between Rop1p and a second protein of 50 kDa whose identity has not yet been confirmed.

Labbe J-C et al. (1999) Biochem Cell Biol "Assessing the function of the Ro ribonucleoprotein complex using Caenorhabditis elegans as a biological tool."

Labbe J-C, Hekimi S, Rokeach LA

[Biochem Cell Biol, 1999]

The Ro ribonucleoprotein complex (Ro RNP) was initially described as an autoimmune target in human diseases such as systemic lupus erythematosus and Sjogren's syndrome. In Xenopus and human cells, its general structure is composed of one major protein of 60 kDa, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Although no function has been assigned to the Ro RNP, Ro60 has been shown to bind mutant 5S ribosomal RNA (rRNA) molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Ro60 has also been shown to participate in the regulation of the translational fate of the L4 ribosomal protein mRNA by interacting with the 5' untranslated region, again suggesting its possible implication in ribosome biogenesis. To identify the function of Ro RNP, we have taken a genetic approach in the nematode Caenorhabditis elegans. As such, we characterized the gene encoding the protein ROP-1, the homologue of the human Ro60 protein. Here, we review the phenotypic analysis of C. elegans rop-l(-) mutants and integrate these results into a model for the function of the Ro RNP particle.

Labbe J-C et al. (2000) West Coast Worm Meeting "Regulation of C. elegans dauer formation by an RNA quality control pathway component"

Labbe J-C, Burgess J, Hekimi S

[West Coast Worm Meeting, 2000]

Under conditions that do not favor developmental growth and reproduction, worms can enter an alternate third-larval stage termed the dauer stage. Dauer larvae are developmentally arrested and are adapted for long term survival and dispersal. In order to make this decision, animals monitor a variety of chemosensory cues including the concentration of a constitutively secreted pheromone, as well as food and temperature. It is currently unknown whether internal states, such as the amount of molecular damage accumulated, is also taken into account when making the decision to enter the dauer stage. The gene rop-1 encodes the worm homologue of the human Ro ribonucleoprotein 60-kDa constituent, which has previously been shown to participate in the quality control of 5S ribosomal RNA. Here, we present biochemical evidence that rop-1 is cleaved at the L2/L3 molt concommittant with entry into L3 but is not cleaved when animals enter the dauer stage. In addition, rop-1 is not cleaved in daf-2 mutants suggesting cleavage of rop-1 may require functional insulin-like signaling. Furthermore, we provide evidence that rop-1 genetically interacts with the insulin receptor-like kinase daf-2 in an allele-specific manner as well as with the TGF-b transforming growth factor daf-7 in a temperature dependent manner. Finally, we show that the enhancement of dauer formation by rop-1 in daf-2(e1370) is daf-16 dependent. However, the processing of rop-1 appears to be daf-16 independent. We suggest that an RNA quality control checkpoint may exist for dauer formation.

Labbe J-C et al. (1995) Gene "The Caenorhabditis elegans rop-1 gene encodes the homologue of the human 60-kDa Ro autoantigen."

Labbe J-C, Jannatipour M, Rokeach LA

[Gene, 1995]

As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (Ro60) in Caenorhabditis elegans (Ce). This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns. The encoded protein, Rop1p, shares 40% identity and 63% overall similarity with both the human and amphibian Ro60. Recombinant protein has been produced in Escherichia coli and used to elicit anti-Rop1p antibodies. Immunological analysis indicated that the Ro60 epitopes have been poorly conserved. Gene-fusion expression studies in transgenic nematodes will provide a new avenue of research to shed

Labbe J-C et al. (1999) Genetics "The levels of the RoRNP-associated Y RNA are dependent upon the presence of ROP-1, the Caenorhabditis elegans Ro60 protein."

Labbe J-C, Hekimi S, Rokeach LA

[Genetics, 1999]

The Ro ribonucleoproteins (RoRNP) consist of at least one major protein of 60 kD, Ro60, and one small associated RNA, designated Y RNA. Although RoRNP have been found in all vertebrate species examined so far, their function remains unknown. The Caenorhabditis elegans rop-1 gene previously has been identified as encoding a Ro60 homologue. We report here the phenotypic characterization of a C. elegans strain in which rop-1 has been disrupted. This is the first report regarding the inactivation of a major RoRNP constituent in any organism. The rop-1 mutant worms display no visible defects. However, at the molecular level, the disruption of rop-1 results in a dramatic decrease in the levels of the ROP-1-associated RNA (CeY RNA). Moreover, transgenic expression of wild-type rop-1 partially rescues the levels of CeY RNA. Considering that transgenes are poorly expressed in the germline, the fact that the rescue is only partial is most likely related to the high abundance of the CeY RNA in the adult germline and in embryos. The developmental expression pattern and localization of CeY RNA suggest a role for this molecule during embryogenesis. We conclude that, under laboratory culture conditions, ROP-1 does not play a crucial role in C. elegans.