Many families of C. elegans microRNAs are not essential for development or viability

Abstract

MicroRNAs (miRNAs) are approximately 23 nt regulatory RNAs that posttranscriptionally inhibit the functions of protein-coding mRNAs. We previously found that most C. elegans miRNAs are individually not essential for development or viability and proposed that paralogous miRNAs might often function redundantly. To test this hypothesis, we generated mutant C. elegans strains that each lack multiple or all members of one of 15 miRNA families. Mutants for 12 of these families did not display strong synthetic abnormalities, suggesting that these miRNA families have subtle roles during development. By contrast, mutants deleted for all members of the mir-35 or mir-51 families died as embryos or early larvae, and mutants deleted for four members of the mir-58 family showed defects in locomotion, body size, and egg laying and an inability to form dauer larvae. Our findings indicate that the regulatory functions of most individual miRNAs and most individual families of miRNAs related in sequence are not critical for development or viability. Conversely, because in some cases miRNA family members act redundantly, our findings emphasize the importance of determining miRNA function in the absence of miRNAs related in sequence.

Figure 1. mir-35 family sequences and mutant phenotypes

(A) Sequences of the eight mir-35 family members. Nucleotides are shown in white letters on black background if more than half of the family members are identical. (B) Mutations in the mir-35 family result in embryonic or L1 lethality. n>200 for each genotype. All experiments were conducted at 20°C except where noted. (C) mir-35-41(nDf50) mir-42(nDf49) mutant animals develop abnormally slowly and arrest during embryogenesis. Timepoints are from time-lapse recordings of individual embryos. Time = 0 min corresponds to the first cell division. In the mir-35-41(nDf50) mir-42(nDf49) embryo the anterior pharynx (white arrowhead) is not attached to the mouth hypodermal cells that normally link the digestive tract to the hypodermis surrounding the embryo (black arrowhead). The wild-type embryo hatched at 654 min. Scale bar, 10 μm. (D) Transgenic expression of any mir-35 family member rescues the embryonic lethality of mir-35-41(nDf50) mir-42(nDf49) animals. We injected mir-35-41(nDf50) mir-42(nDf49)/mIn1[mIs14 dpy-10(e128)] animals with constructs encoding single miRNAs and established transgenic lines. We concluded that a transgene had rescued the lethality if we could maintain a homozygous mir-35-41(nDf50) mir-42(nDf49) strain carrying the transgene. (E) Scatter plot of the normalized intensities from a miRNA microarray comparing fem-3(q20ts) to fem-2(b245ts) expression. The germline of fem-3(q20ts) animals produces only sperm, while the germline of fem-2(b245ts) animals produces only oocytes at the restrictive temperature. See also Figure S1.

Figure 2. mir-51 family sequences and mutant phenotypes

(A) Sequences of the C. elegans (C.e.) mir-51 family members and their human (H.s.) homologs. Nucleotides are shown in white letters on black background if more than half of the C. elegans family members are identical. (B) Some mir-51 family mutant combinations result in slow growth and embryonic or early larval lethality. The percent of animals that had progressed beyond the late fourth larval (L4) stage 48 h or 72 h after they were laid as embryos is indicated; n>100 for each genotype and timepoint. Table S9 lists the genotypes of the strains scored. (C) Animals lacking all mir-51-56 family members (mir-51 mir-53(nDf67); mir-52(n4114); mir-54-56(nDf58)) died as embryos or early larvae. Micrographs were taken 16-18 h after the first cell division. Scale bars, 10 μm. In the mir-51 mir-53(nDf67); mir-52(n4114); mir-54-56(nDf58) embryo the anterior pharynx (white arrowhead) is not attached to the hypodermal cells of the mouth that normally link the digestive tract to the hypodermis surrounding the embryo (black arrowhead). The newly hatched larva shows gross posterior body abnormalities. (D) Expression of single mir-51 family members rescues the embryonic lethality caused by lack of all members. We injected heterozygote mir-51 mir-53(nDf67) mir-52(n4114)/nT1 [qIs51]; mir-54-56(nDf58) animals with constructs containing single miRNAs and established transgenic lines. We concluded that a transgene had rescued the lethality if we could maintain a homozygous mir-51 mir-53(nDf67) mir-52(n4114); mir-54-56(nDf58) strain carrying the transgene. See also Figure S2.

Figure 3. mir-58 family sequences and mutant phenotypes

(A) Sequences of the C. elegans (C.e.) mir-58 family and the Drosophila melanogaster (D.m.) homolog bantam. Nucleotides are shown in white letters on black background if more than half of the C. elegans family members are identical. (B) mir-80(nDf53); mir-58(n4640); mir-81-82(nDf54) mutants are smaller than the wild type. A transgene carrying mir-80 (Ex[mir-80]) rescued the small body size defect of mir-80(nDf53); mir-58(n4640); mir-81-82(nDf54) mutants. Micrographs were taken 48 h after the late L4 (fourth larval) stage. Scale bars, 100 μm. (C) mir-58 family mutants lay late-stage embryos. Distributions of the developmental stages of eggs laid by wild-type animals, single and combinations of mir-58 family mutants, and mir-58,80,81,82 animals carrying a mir-80 transgene (Ex[mir-80]). At least 100 embryos were scored for each genotype. (D) mir-58 family mutants are defective in the formation of dauer larvae. “YES”, dauers were isolated from every plate assayed; “NO”, no dauers were isolated from any of the plates assayed. n, number of plates. Two independently isolated transgenic lines were tested for the mir-82 and T07D1.2::mir-82 transgenes (three plates for each line), and one line was tested for each of the other transgenes. mir-82 is contained within an intron of T07D1.2. The transgene designated [mir-80Δ] had the sequence corresponding to mir-80 deleted. See also Figure S3.