Integral Nuclear Pore Proteins Bind to Pol III-Transcribed Genes and Are Required for Pol III Transcript Processing in C. elegans

Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-β IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans

Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

Abstract Background Although Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known. Results We determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point. Conclusions Local DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus

Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

Abstract Background Although Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known. Results We determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point. Conclusions Local DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus

Reduced Insulin/IGF-1 Signaling Restores Germ Cell Immortality to Caenorhabditis elegans Piwi Mutants

Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C. elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNA interference mutants with strong transposon silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNA interference proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system

Comparative analysis of metazoan chromatin organization

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosoph ..

Nucleoporins Nup153 and Mtor are located at both NPCs and the nuclear interior, and associate with active transcription.

<p>(A) Schematic representation of Nup153 (left) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000861#pgen.1000861-Rabut1" target="_blank">[13]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000861#pgen.1000861-Ball1" target="_blank">[15]</a> and Mtor (right) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000861#pgen.1000861-Krull1" target="_blank">[14]</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000861#pgen.1000861-Zimowska1" target="_blank">[16]</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000861#pgen.1000861-Zimowska2" target="_blank">[17]</a> localization at the NPC and the nucleoplasm. <i>Nup153</i> is proposed to be localized at the nuclear coaxial ring in proximity to the nuclear membrane (1); at the distal pore basket (2); as nucleoplasmic filaments (3); and shuttle between NPCs and the nucleoplasmic pool (arrow, 4). <i>Mtor</i> is proposed to constitute the pore basket (5) and nucleoplasmic filaments or granules (6). The mobile property of Mtor is unknown (7). It is still unclear whether the nucleoplasmic NUP153 and Mtor structures are extended from NPCs (dotted ovals). (B) Possible role of nucleoplasmic nucleoporins in transporting mRNA from the nuclear interior to NPCs.</p

Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.[Background]: Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components. [Results]: Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions. [Conclusions]: We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.This work was funded by the Spanish Ministry of Science and Innovation (BFU-2010-15478 to PA), and the European Regional Development Fund. Some nematode strains used in this work were provided by the National Bioresource Project for the Nematode C. elegans (directed by Shohei Mitani), the International C. elegans Gene Knockout Consortium, and the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis, MN), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).Peer Reviewe