High-throughput profiling of caenorhabditis elegans starvation-responsive microRNAs

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-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr 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

Expression of selected miRNAs and mRNAs involved in the fasting response as quantified by qRT-PCR.

<p>Relative quantification of (A) miR-240-5p, miR-246-3p, and (B) miR-35-3p, miR-36-3p, miR-39-3p, and <i>gld-1</i> and <i>lin-23</i> mRNA abundance in well-fed and 12-hr starved early L4 larvae by qRT-PCR. For miR-240-5p, miR-246-3p, miR-35-3p, miR-36-3p, miR-39-3p quantification, miR-58-3p was used as a control. For <i>gld-1</i> and <i>lin-23</i> mRNAs quantification, <i>β-actin</i> mRNA was used as a control. Error bars represent the standard error of the mean of three independent experiments. (miR-240-5p) p = 0.03, (miR-246-3p) p = 0.03, (miR-35-3p, SEM = 37.60) p = 0.03, (miR-36-3p, SEM = 14.33) p = 0.03, (miR-39-3p) p = 0.03, (<i>gld-1</i>, SEM = 20.87) p = 0.03 and (<i>lin-23</i>, SEM = 19.74) p = 0.4, calculated by the Wilcoxon Signed Rank Test.</p

Functional classification of sRNA-seq reads.

<p>Reads were classified according to the type of sequence to which they mapped (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142262#sec002" target="_blank">Materials and Methods</a>). (A) Well-fed, and (B) 12-hr starved early L4 larvae.</p

Kaplan-Meir survival curves of well-fed (n = 117) and 12-hr starved (n = 112) early L4 larvae.

<p>Starved worms showed a significantly increased lifespan (*) compared to well-fed early L4 larvae, according to a log-rank (Mantel-Cox) test (<i>p</i> < 0.0001).</p

Physical aspect, length and lipid content of well-fed and 12-hr starved early L4 larvae.

<p>(A) Length of well-fed and 12-hr starved early L4 larvae, n = 30 early L4 larvae per group, mean ± standard error of the mean, p < 0.0001 according to a Wilcoxon Signed Rank Test. (B) Lipid content of well-fed and 12-hr starved early L4 larvae stained with Oil-Red-O (20X objective).</p

Differential expression analysis of miRNAs that changed their expression in 12-hr starved compared to well-fed early L4 larvae.

<p>MA-plot showing the absolute and relative expression of all known miRNAs with at least 1 read per million in one of the two libraries (well-fed and 12-hr starved worms). Positive (negative) log₂ fold-changes represent miRNAs with higher (lower) expression under fasting conditions. Significantly differentially expressed miRNAs (False Discovery Rate <5%) are shown as red circles. Grey text indicates three miRNA hairpins (excluding annotated mature products) also found to be differentially expressed.</p

The most abundant miRNAs in each library.

<p>The most abundant miRNAs in well-fed and 12-hr starved early L4 larvae are represented as the percentage of reads that mapped to known miRNAs.</p