[
International Worm Meeting,
2017]
Nuclear Enriched Abundant Transcript 1 (NEAT1) is a long non-coding RNA necessary for the formation of nuclear bodies called paraspeckles (composed of about 40 proteins) in mammals. NEAT1 is expressed as two isoforms (3.7kb and 23kb). This transcript has been implicated in different neurodegenerative diseases (including ALS/FTD and MS), brain viral infection (Japanese encephalitis virus and rabies virus), in addition to playing an oncogenic role in most solid tumors. In regards to ALS, it has been shown that NEAT1 is directly bound by TDP-43 and that its expression is highly up-regulated in human motor neurons presenting ALS pathology. In our transcriptome analysis of TDP-1 knock-out and FUS knock-out C. elegans we noticed a significant overexpression of a transcript (C30E1.9), which presents itself in two isoforms of length ~4kb and 23kb. We hypothesized that this transcript was the NEAT1 ortholog in nematodes. We performed smRNA FISH experiments and observed the formation of nuclear bodies in both N2 and TDP-1 knock-out worms: no statistical significance was noticed in the number of nuclear bodies between the two strains. As is the case for NEAT1 in mammals, we did not observe this transcript in the germline. We performed smRNA FISH in strains expressing human TDP-43::GFP and TDP-1::GFP in order to determine whether the lncRNA C30E1.9 co-localizes with TDP-43 and TDP-1: no strong co-localization was found. Feeding RNAi against the C30E1.9 transcript has so far been inconclusive, as no phenotypes have been observed, but clear reduction in the C30E1.9 nuclear bodies has not been observed. Ultimately, given that NONO protein in mammals is required for NEAT1 driven paraspeckle nucleation, we performed smRNA FISH in a NONO-1 (C. elegans ortholog of human NONO) knock-out balancer strain: we found no evidence of reduced number of nuclear bodies between NONO-1 knock-out and NONO-1 expressing strains. We conclude that C30E1.9 is most likely not a NEAT1 ortholog, but it could potentially play an important regulatory function in C. elegans given its expression pattern, the formation of nuclear bodies, and its greatly elevated expression levels in C. elegans ALS disease model.
[
International Worm Meeting,
2005]
We generated a C. elegans interactome map that contains ~5,500 potential interactions, referred to as Worm Interactome version 5 (WI5) (Li et al. Science 2004). Together with another interactome map for Drosophila melanogaster, these datasets represent the first of their kind for metazoan organisms. Although already helpful, the protein interaction data in WI5 is far from complete and needs improvement. Here we describe the initiation of a new approach for the generation of a worm interactome version 6 (WI6). The first challenge of the WI6 project consisted in the generation of a worm ORFeome resource referred to as the worm ORFeome version 1. This collection of ~11,000 cloned ORFs represents a useful platform for the application of reverse proteomic approaches for worm. The WI6 project uses the worm ORFeome version 1 as a starting point, and is designed to cover three fundamental aspects of the interactome map: completeness, coverage, and quality. The second challenge was the development of an improved version of the yeast two-hybrid system. The goal of the WI6 project is to test all 11,000 cloned ORFs by yeast two-hybrid (Y2H) (1,21 x 108 pairwise combinations). We devised and validated a new protocol that combines both efficient HT settings and high level of saturation. The production phase of the project was recently initiated. We are able to test ~4 x 106 pairwise combinations per week. So far, we tested ~10% of the total matrix and found ~400 novel Y2H interactions. We expect to map an additional ~4,000 interactions by the end of the WI6 project. Successive interactome versions depend upon improvements in genome annotation and the progress of the ORFeome cloning project. Gradually our goal will be to expand the worm interactome map into more complete versions. Our ORFeome project has now evolved into a version 3.1 (see Abstract by Lamesch et al.), from which approximately 2,000 additional ORFs have been cloned. Ultimately, the aim is to generate a high quality interactome map that, together with other functional genomic and proteomic (or omic) information (Vidal Cell 2001), will serve as a backbone for the drawing of a global functional wiring diagram. The work in progress will be presented.