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. 2015 Jun 11:5:11284.
doi: 10.1038/srep11284.

microRNA regulation of the embryonic hypoxic response in Caenorhabditis elegans

Konstantinos Kagias  1 , Roger Pocock  2
Affiliations

microRNA regulation of the embryonic hypoxic response in Caenorhabditis elegans

Konstantinos Kagias et al. Sci Rep. .

Abstract

Layered strategies to combat hypoxia provide flexibility in dynamic oxygen environments. Here we show that multiple miRNAs are required for hypoxic survival responses during C. elegans embryogenesis. Certain miRNAs promote while others antagonize the hypoxic survival response. We found that expression of the mir-35 family is regulated by hypoxia in a HIF-1-independent manner and loss of mir-35-41 weakens hypoxic survival mechanisms in embryos. In addition, correct regulation of the RNA binding protein, SUP-26, a mir-35 family target, is needed for survival in chronic hypoxia. The identification of the full mRNA target repertoire of these miRNAs will reveal the miRNA-regulated network of hypoxic survival mechanisms in C. elegans.

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Figures

Figure 1
Figure 1. The mir-35 family is required for embryonic hypoxic survival.
(A) mir-35–41 locus, mutant alleles and genomic rescue fragment. nDf50 (blue) and gk262 (red) alleles remove the entire mir-35–41 locus and part of the Y62F5A.9 gene. The genomic rescue fragment used in (D) is marked in green, which includes a 602 bp upstream region and the mir-35 hairpin. (B) At 21% O2, two mir-35–41 mutant alleles, nDf50 and gk262, exhibit 50% and 75% embryonic lethality respectively, whereas, wild type and hif-1(ia4) mutant embryos exhibit minimal lethality. At 0.5% O2, both mir-35–41 mutants approach 100% embryonic lethality (n = 176–242), whereas wild type and hif-1(ia4) mutant embryos exhibit 10% (n = 974) and 40% (n = 1132) lethality respectively. These data partially overlap with Table S1. (C) An increased percentage of nDf50 mutant embryos die younger in hypoxia than in normoxia. ‘Malformed’ refers to embryos with severe defects in their overall structure that do not permit stage identification. (D) Normoxic and hypoxic lethality of nDf50 mutant embryos is rescued by transgenic expression of mir-35. The sequence used to rescue mir-35 is shown as a green line in (A). n = 96–125. # refers to independent transgenic lines. Contingency table values are presented and Fischer exact test applied for statistical evaluation. ***≤0.001, ****≤0.0001. n.s. = not significant.
Figure 2
Figure 2. The mir-35–41 promoter drives ubiquitous expression in the embryo.
A mir-35–41prom::2xNLS::yfp transcriptional reporter drives expression throughout the embryo from the 20 cell stage. The region used to drive yfp expression is shown in yellow on the genomic view (top). Upper panels are Nomarski micrographs and bottom panels are fluorescence images of the same embryos. Anterior is to the left. Scale bar 10 μm.
Figure 3
Figure 3. Expression of the mir-35 family is regulated by chronic hypoxia.
(A–B) qRT-PCR showing mir-35 family member expression levels in wild type embryos exposed to 21% O2 (black bars) or 0.5% O2 (orange bars) for 20 mins (A) or 4 hrs (B). The level of normoxic expression was set to 1 for each of the three repetitions. (C) Graphical representation of mir-35 family member abundance in normoxia. mir-38 and mir-41 are less abundant than the other family members, even from those miRNAs transcribed in the same cluster. Values on the graph are logarithmic functions with base 10 of the fold change value for each miRNA. mir-41 showed the lowest relative abundance and was arbitrarily set as the value 1. Primer efficiencies are 116%, 118%, 116%, 113%, 93%, 93%, 109% and 118% for each respective miRNA. (D) Intensity of GFP expression driven by the mir-35–41 promoter is unaffected by 4 hrs of hypoxic exposure. The transgene used is wwIs8[pmir-35–41::GFP + unc-119(+)]. (E–F) gfp transcription, driven by the mir-35–41 promoter in wild type embryos exposed to 21% O2 (black bars) or 0.5% O2 (orange bars) for 20 mins (E) or 4 hrs (F). Data are presented as means of at least 3 independent repetitions and error bars represent ± SD. Students t-test was used to assess for statistical significance. *p ≤ 0.05, **≤0.01, ***p ≤ 0.001, ****≤ 0.0001, n.s. = not significant.
Figure 4
Figure 4. The sup-26 promoter drives ubiquitous expression in the embryo.
A sup-26prom::2xNLS::yfp transcriptional reporter drives expression throughout the embryo from the 80 cell stage. Region used to drive YFP expression is shown in yellow on the genomic view (top). Upper panels are Nomarski micrographs and bottom panels are fluorescence images of the same embryos. Anterior to the left. Scale bar 10 μm.
Figure 5
Figure 5. sup-26 is a mir-35–41 target and is required for hypoxic survival.
(A) The C. elegans sup-26 3’ UTR contains a single mir-35 family binding site (only mir-35 is shown). The mir-35 seed sequence is shown in blue and the predicted sup-26 3’UTR is shown in red. (B) Sensor experiment constructs. mir-35 was expressed in the pharynx together with a RFP reporter controlled by the unregulated unc-54 3’UTR and a GFP reporter controlled by the sup-26 3’UTR (wild type or mir-35 binding site mutated). The mir-35 binding site in the sup-26 3’UTR was mutated from CCCGGUG to CCatGgG to prevent binding of mir-35 family miRNAs. (C) Representative picture of the sensor experiment results. mir-35 downregulates GFP expression (sup-26 3’UTR) and not the RFP sensor (control 3’UTR). Regulation via the sup-26 3’UTR is dependent on the mir-35 binding site. (D) Quantification of the sensor experiment results. n > 50. Fischer exact test was used for statistical evaluation. # refers to independent transgenic lines. ****≤0.0001.
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