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Spatio-temporal m(i)RNA architecture and 3' UTR regulation in the C. elegans germline

ABSTRACT: In animal germlines, regulation of cell proliferation and differentiation is particularly important but poorly understood. Here, using a cryo-cut approach, we mapped RNA expression along the Caenorhabditis elegans germline and, using mutants, dissected gene regulatory mechanisms that control spatio-temporal expression. We detected, at near single-cell resolution, > 10,000 mRNAs, > 300 miRNAs and numerous unannotated miRNAs. Most RNAs were organized in distinct spatial patterns. Germline-specific miRNAs and their targets were co-localized. Moreover, we observed differential 3’ UTR isoform usage for hundreds of mRNAs. In tumorous gld-2 gld-1 mutants, gene expression was strongly perturbed. In particular, differential 3’ UTR usage was significantly impaired. We propose that PIE-1, a transcriptional repressor, functions to maintain spatial gene expression. Our data also suggest that cpsf-4 and fipp-1 control differential 3’ UTR usage for hundreds of genes. Finally, we constructed a “virtual gonad” enabling “virtual in situ hybridizations” and access to all data (https://shiny.mdc-berlin.de/spacegerm/).

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE115884 | GEO | 2018/11/15

REPOSITORIES: GEO

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Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ÂºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ÂºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300Âµl beads and 150Âµg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ÂºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).

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Spatio-temporal m(i)RNA architecture and 3' UTR regulation in the C. elegans germline

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