Transcriptomic Signature of the Simulated Microgravity Response in Caenorhabditis elegans and Comparison to Spaceflight Experiments.

Given the growing interest in human exploration of space, it is crucial to identify the effects of space conditions on biological processes. Here, we analyze the transcriptomic response of Caenorhabditis elegans to simulated microgravity and observe the maintained transcriptomic response after returning to ground conditions for four, eight, and twelve days. We show that 75% of the simulated microgravity-induced changes on gene expression persist after returning to ground conditions for four days while most of these changes are reverted after twelve days. Our results from integrative RNA-seq and mass spectrometry analyses suggest that simulated microgravity affects longevity-regulating insulin/IGF-1 and sphingolipid signaling pathways. Finally, we identified 118 genes that are commonly differentially expressed in simulated microgravity- and space-exposed worms. Overall, this work provides insight into the effect of microgravity on biological systems during and after exposure.

Effects of liquid cultivation on gene expression and phenotype of C. elegans.

BACKGROUND: Liquid cultures have been commonly used in space, toxicology, and pharmacology studies of Caenorhabditis elegans. However, the knowledge about transcriptomic alterations caused by liquid cultivation remains limited. Moreover, the impact of different genotypes in rapid adaptive responses to environmental changes (e.g., liquid cultivation) is often overlooked. Here, we report the transcriptomic and phenotypic responses of laboratory N2 and the wild-isolate AB1 strains after culturing P0 worms on agar plates, F1 in liquid cultures, and F2 back on agar plates. RESULTS: Significant variations were found in the gene expressions between the N2 and AB1 strains in response to liquid cultivation. The results demonstrated that 8-34% of the environmental change-induced transcriptional responses are transmitted to the subsequent generation. By categorizing the gene expressions for genotype, environment, and genotype-environment interactions, we identified that the genotype has a substantial impact on the adaptive responses. Functional analysis of the transcriptome showed correlation with phenotypical changes. For example, the N2 strain exhibited alterations in both phenotype and gene expressions for germline and cuticle in axenic liquid cultivation. We found transcript evidence to approximately 21% of the computationally predicted genes in C. elegans by exposing the worms to environmental changes. CONCLUSIONS: The presented study reveals substantial differences between N2 and AB1 strains for transcriptomic and phenotypical responses to rapid environmental changes. Our data can provide standard controls for future studies for the liquid cultivation of C. elegans and enable the discovery of condition-specific genes.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

ChIP and Chips: Introducing the WormPharm for correlative studies employing pharmacology and genome-wide analyses in C. elegans.

We present the WormPharm, an automated microfluidic platform that utilizes an axenic medium to culture C. elegans. The WormPharm is capable of sustaining C. elegans for extended periods, while recording worm development and growth with high temporal resolution ranging from seconds to minutes over several days to months. We demonstrate the utility of the device to monitor C. elegans growth in the presence of varying doses of nicotine and alcohol. Furthermore, we show that C. elegans cultured in the WormPharm are amendable for high-throughput genomic assays, i.e. chromatin-immunoprecipitation followed by next generation sequencing, and confirm that nematodes grown in monoxenic and axenic cultures exhibit genetic modifications that correlate with observed phenotypes. The WormPharm is a powerful tool for analyzing the effects of chemical, nutritional and environmental variations on organism level responses in conjunction with genome-wide changes in C. elegans.

Co-translational localization of an LTR-retrotransposon RNA to the endoplasmic reticulum nucleates virus-like particle assembly sites.

The transcript of retrovirus-like transposons functions as an mRNA for synthesis of capsid and replication proteins and as the genomic RNA of virus-like particles (VLPs), wherein the genome is replicated. Retrotransposon RNA and proteins coalesce in a cytoplasmic focus, or retrosome, to initiate VLP assembly, but it is not known how the retrosome is nucleated. We determined how the RNA and Gag protein of the Saccharomyces cerevisiae Ty1 retrotransposon are directed to the retrosome. We found that Ty1 RNA is translated in association with signal recognition particle (SRP), a universally conserved chaperone that binds specific ribosome-nascent chain (RNC) complexes and targets the nascent peptide to the endoplasmic reticulum (ER). Gag is translocated to the ER lumen; yet, it is also found in the cytoplasm, associated with SRP-RNC complexes. In the absence of ER translocation, Gag is synthesized but rapidly degraded, and Ty1 RNA does not coalesce in retrosomes. These findings suggest that Gag adopts a stable conformation in the ER lumen, is retrotranslocated to the cytoplasm, binds to Ty1 RNA on SRP-RNC complexes and multimerizes to nucleate retrosomes. Consistent with this model, we show that slowing the rate of co-translational ER translocation by limiting SRP increases the prevalence of retrosomes, while suppressing the translocation defect of srp hypomorphs by slowing translational elongation rapidly decreases retrosome formation. Thus, retrosomes are dynamic foci of Ty1 RNA-RNC complexes whose formation is modulated by the rate of co-translational ER translocation. Together, these findings suggest that translating Ty1 mRNA and the genomic RNA of VLPs originate in a single pool and moreover, that co-translational localization of Ty1 RNA nucleates the presumptive VLP assembly site. The separation of nascent Gag from its RNA template by transit through the ER allows Gag to bind translating Ty1 RNA without displaying a cis-preference for its encoding RNA.

C. elegans RNA-binding protein GLD-1 recognizes its multiple targets using sequence, context, and structural information to repress translation.

Caenorhabditis elegans GLD-1, a maxi-KH motif containing RNA-binding protein, has various functions mainly during female germ cell development, suggesting that it likely controls the expression of a selective group of maternal mRNAs. To gain an insight into how GLD-1 specifically recognizes these mRNA targets, we identified 38 biochemically proven GLD-1 binding regions from multiple mRNA targets that are among over 100 putative targets co-immunoprecipitated with GLD-1. The sequence information of these regions revealed three over-represented and phylogenetically conserved sequence motifs. We found that two of the motifs, one of which is novel, are important for GLD-1 binding in several GLD-1 binding regions but not in other regions. Further analyses indicate that the importance of one of the sequence motifs is dependent on two aspects: (1) surrounding sequence information, likely acting as an accessory feature for GLD-1 to efficiently select the sequence motif and (2) RNA secondary structural environment where the sequence motif resides, which likely provides "binding-site accessibility" for GLD-1 to effectively recognize its targets. Our data suggest some mRNAs recruit GLD-1 by a distinct mechanism, which involves more than one sequence motif that needs to be embedded in the correct context and structural environment.