Effect of life history on microRNA expression during C. elegans development

Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs after the second larval stage in response to environmental stimuli encountered during the first two larval stages. Dauer larvae are stress resistant and long lived, permitting survival in harsh conditions. When environmental conditions improve, dauer larvae re-enter development, and progress through two post-dauer larval stages to adulthood. Strikingly, all of these life history options (whether continuous or interrupted) involve an identical pattern and sequence of cell division and cell fates. To identify microRNAs with potential functions in buffering development in the context of C. elegans life history options, we used multiplex real-time PCR to assess the expression of 107 microRNAs throughout development in both continuous and interrupted life histories. We identified 17 microRNAs whose developmental profile of expression is affected by dauer life history and/or L1 diapause, compared to continuous development. Hence these microRNAs could function to regulate gene expression programs appropriate for different life history options in the developing worm

Mutations in Conserved Residues of the C. elegans microRNA Argonaute ALG-1 Identify Separable Functions in ALG-1 miRISC Loading and Target Repression

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition

How to minimise the effect of tumour cell content in detection of aberrant genetic markers in neuroblastoma

Background:Clinical heterogeneity reflects the complexity of genetic events associated with neuroblastoma (NB). To identify the status of all described genetic loci with possible prognostic interest, high-throughput approaches have been used, but only with tumour cell content >60%. In some tumours, necrotic, haemorrhagic and/or calcification areas influence the low amount of neuroblasts. We evaluated the effect of tumour cell content in the detection of relevant aberrant genetic markers (AGM) diagnosed by fluorescence in situ hybridisation (FISH) on tissue microarrays (TMA) in NB.Methods:Two hundred and thirty-three MYCN non-amplified primary NB included in 12 TMAs were analysed.Results:Presence of AGM reduced event-free survival (EFS) (P=0.004) as well as overall survival (OS) (P=0.004) of patients in the whole cohort. There were no differences in prognostic impact of presence of AGM according to tumour cell content.Conclusion:We propose the use of FISH to diagnose AGM of all NB samples having the above-mentioned areas to determine patient risk

Most \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e MicroRNAs are Individually Not Essential for Development or Viability

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs

The Pseudomonas aeruginosa accessory genome elements influence virulence towards Caenorhabditis elegans

BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships. RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence. CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance

Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project

Background: In the INRG dataset, the hypothesis that any segmental chromosomal alteration might be of prognostic impact in neuroblastoma without MYCN amplification (MNA) was tested. Methods: The presence of any segmental chromosomal alteration (chromosome 1p deletion, 11q deletion and/or chromosome 17q gain) defined a segmental genomic profile. Only tumours with a confirmed unaltered status for all three chromosome arms were considered as having no segmental chromosomal alterations. Results: Among the 8800 patients in the INRG database, a genomic type could be attributed for 505 patients without MNA: 397 cases had a segmental genomic type, whereas 108 cases had an absence of any segmental alteration. A segmental genomic type was more frequent in patients >18 months and in stage 4 disease (P<0.0001). In univariate analysis, 11q deletion, 17q gain and a segmental genomic type were associated with a poorer event-free survival (EFS) (P<0.0001, P=0.0002 and P<0.0001, respectively). In multivariate analysis modelling EFS, the parameters age, stage and a segmental genomic type were retained in the model, whereas the individual genetic markers were not (P<0.0001 and RR=2.56; P=0.0002 and RR=1.8; P=0.01 and RR=1.7, respectively). Conclusion: A segmental genomic profile, rather than the single genetic markers, adds prognostic information to the clinical markers age and stage in neuroblastoma patients without MNA, underlining the importance of pangenomic studies

Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy

[EN] Viroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg2+ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg2+ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) grants BIO2016-79618-R (funded by EU under the FEDER programme) to C.B. and BFU2104-56812-P to R.F., as well as by the Comunidad de Madrid grant S2018/NMT-4349 to L.V. 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International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee

Neuroblastoma serves as a paradigm for utilising tumour genomic data for determining patient prognosis and treatment allocation. However, before the establishment of the International Neuroblastoma Risk Group (INRG) Task Force in 2004, international consensus on markers, methodology, and data interpretation did not exist, compromising the reliability of decisive genetic markers and inhibiting translational research efforts. The objectives of the INRG Biology Committee were to identify highly prognostic genetic aberrations to be included in the new INRG risk classification schema and to develop precise definitions, decisive biomarkers, and technique standardisation. The review of the INRG database (n=8800 patients) by the INRG Task Force finally enabled the identification of the most significant neuroblastoma biomarkers. In addition, the Biology Committee compared the standard operating procedures of different cooperative groups to arrive at international consensus for methodology, nomenclature, and future directions. Consensus was reached to include MYCN status, 11q23 allelic status, and ploidy in the INRG classification system on the basis of an evidence-based review of the INRG database. Standardised operating procedures for analysing these genetic factors were adopted, and criteria for proper nomenclature were developed. Neuroblastoma treatment planning is highly dependant on tumour cell genomic features, and it is likely that a comprehensive panel of DNA-based biomarkers will be used in future risk assignment algorithms applying genome-wide techniques. Consensus on methodology and interpretation is essential for uniform INRG classification and will greatly facilitate international and cooperative clinical and translational research studies