High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

RESEARCH ARTICLE High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs Laura Garcia-Segura1,2, Cei Abreu-Goodger3, Armando Hernandez-Mendoza4, Tzvetanka D. Dimitrova Dinkova5, Luis Padilla-Noriega6, Martha Elva Perez-Andrade2, Juan MirandaRios2* 1 Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), México, D.F., México, 2 Unidad de Genética de la Nutrición, Depto. de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM e Instituto Nacional de Pediatría, México, D.F., México, 3 Unidad de Genómica Avanzada (Langebio), CINVESTAV, Irapuato, Guanajuato, México, 4 Centro de Investigación en Dinámica Celular, Universidad Autónoma del Edo. de Morelos, Cuernavaca, Morelos, México, 5 Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México, D.F., México, 6 Departamento de Virología, Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F., México * OPEN ACCESS Citation: Garcia-Segura L, Abreu-Goodger C, Hernandez-Mendoza A, Dimitrova Dinkova TD, Padilla-Noriega L, Perez-Andrade ME, et al. (2015) High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs. PLoS ONE 10 (11): e0142262. doi:10.1371/journal.pone.0142262 Editor: Partha Mukhopadhyay, National Institutes of Health, UNITED STATES Received: July 8, 2015 Accepted: October 20, 2015 Published: November 10, 2015 Copyright: © 2015 Garcia-Segura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available in the manuscript, its Supporting Information Files, and the NCBI Gene Expression Omnibus (GEO) database under accession number GSE67711. Funding: Funding was provided by Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica (PAPIIT), Direccion General de Asuntos del Personal Academico (DGAPA), Universidad Nacional Autonoma de Mexico, grants IN209310-3 and IN203514, dgapa.unam.mx, and Fondos Federales Instituto Nacional de Pediatria, grant 036/ Abstract MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-48135p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans. PLOS ONE | DOI:10.1371/journal.pone.0142262 November 10, 2015 1 / 22 C. elegans Starvation-Responsive microRNAs 2015. This paper presents part of the PhD thesis of Laura García-Segura, who is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and received fellowship 384014 from Consejo Nacional de Ciencia y Tecnología (CONACYT), México. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction When cells are deprived of nutrients, they respond to starvation by changes in intracellular signaling, in order to enhance their chances of survival [1]. One such response is metabolism modulation by activating catabolic pathways and suppressing anabolic ones, generating necessary metabolites to maintain core cellular activities [2]. If homeostasis cannot be re-established, a new gene expression program is enforced to try to escape cell death. MicroRNAs (miRNAs) are thought to help maintain homeostasis and/or reprogram gene expression [3]. miRNAs are non-coding, short RNAs of approximately ~22 bases that modulate stability and translational capacity of their mRNA targets [4]. More than half of all mammalian mRNAs are predicted targets of miRNAs [5]. Considering that the number of human miRNAs already reported is 2,603 (miRBase Release 20 [6]), and maybe reaching over six thousand, according to a recent analysis of sRNA-seq datasets from 13 human tissue types [7], they outnumber cellular kinases and phosphatases (more than 500 kinases and 150 phosphatases have been predicted in the human proteome), emphasizing their importance in regulation. MiRNAs also play key roles in mediating stress responses [8]. Paradoxically, inactivation of most individual miRNAs in flies and worms has no effect on viability or development when assayed under standard laboratory conditions [9–11]. In contrast, they seem to be indispensable when mutant animals are subjected to stress conditions: e.g. miR-7 knockout flies are unable to properly develop their eyes if the flies are housed in an environment that shows cyclic temperature variations [12]. Stress conditions can produce dramatic changes in miRNA biogenesis, subcellular localization, activities of miRNA-protein complexes and the expression of their targets [3]. Several fundamental phenomena were discovered in C. elegans such as programmed cell death, RNAi and endogenous regulation by miRNAs. C. elegans is a relatively simple animal formed by 959 somatic cells in the hermaphrodite and is widely used as an experimental model due to the large amount of genetic tools and available mutants. It has a short generation time, is easy to culture and has a fully sequenced genome. Because it is completely transparent, it allows direct visualization of gene expression with repor (...truncated)