-
[
Science,
2005]
The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. We identified two PER1-associated factors, NONO and WDR5, that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuated circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele were nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augmented PER-mediated transcriptional repression, and its reduction by RNAi diminished circadian histone methylations at the promoter of a clock gene.
-
[
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.
-
[
Protein Sci,
2015]
Members of the Drosophila behaviour/human splicing (DBHS) protein family have been characterised in the vertebrates Homo sapiens and Mus musculus, and the invertebrates Drosophila melanogaster and Chironomus tentans. Collectively, both vertebrate and invertebrate DBHS proteins function throughout gene regulation, largely but not always, within the nucleus. In this study, we report a structural and bioinformatic analysis of the DBHS protein family to guide future studies into DBHS protein function. To explore the structural plasticity of the family, we describe the 2.4-A-crystal structure of Caenorhabditis elegans NONO-1. The structure is dimeric, with a domain arrangement consistent with mammalian DBHS proteins. Comparison with the DBHS structures available from H. sapiens reveals that there is inherent domain flexibility within the homologous DBHS region. Mapping amino acid similarity within the family to the NONO-1 dimer highlights the dimer interface, coiled-coil oligomerisation motif and putative RNA binding surfaces. Surprisingly, the interior surface of RRM2 that faces a large internal void is highly variable, but the external 2-3 loops of RRM2 show remarkable preservation. Overall, the DBHS region is under strong purifying selection whereas the sequences N- and C- terminal to the DBHS region are less constrained. The findings described in this study provide a molecular basis for further investigation into the mechanistic function of the DBHS protein family in biology. This article is protected by copyright. All rights reserved.
-
[
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.
-
[
Neuron,
2004]
RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.