Functional lability of RNA-dependent RNA polymerases in animals

Abstract

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways.

Fig 1. Phylogenetic distribution of RdRP genes in metazoans.

A. Proteome sequences from 538 metazoans were screened for potential RdRPs. For each clade indicated on the right edge, n is the number of species analyzed in the clade, and piecharts indicate the proportion of species possessing RdRP genes (with each RdRP family represented by one piechart, according to the color code given at the top left). B. An HMMer search identifies 6 candidate RdRPs in the predicted Branchiostoma lanceolatum proteome. Only 2 candidates have a complete RdRP domain (represented by a red bar with round ends; note that apparent domain truncations may be due to defective proteome prediction). A white star indicates that every catalytic amino acid is present. Candidate BL02069 also possesses an additional known domain, AAA_12 (in yellow).

Fig 2. Eukaryotic RdRP phylogeny supports the vertical transfer scenario.

A. Bayesian phylogenetic tree of the eukaryotic RdRP family. α, β and γ clades of eukaryotic RdRPs have been defined by [31]. Sectors highlighted in grey are detailed in panels B, C and D for clarity. Scale bar: 0.4 amino acid substitution per position. Posterior probability values are indicated for each node in panels B–D.

Fig 3. Detection of B. lanceolatum small RNAs.

A. Four libraries were prepared for each biological sample, to detect small RNAs bearing either a single 5′ phosphate (Libraries #1 and 2) or any other number of phosphates (including zero; Libraries #3 and 4), and either a (2′-OH and 3′-OH) or a protected 3′ end (Libraries #1 and 3), or specifically a protected (e.g., 2′-O-methylated) 3′ end (Libraries #2 and 4). hpf: hours post fertilization. B. Size distribution of genome-matching adult male small RNAs, excluding reads that match abundant non-coding RNAs (rRNAs, tRNAs, snRNAs, snoRNAs or scaRNAs). Read numbers are normalized by the total number of genome-matching reads (including <18 nt and >30 nt reads) that do not match abundant non-coding RNAs, and expressed as parts per million (ppm). C. Size distribution of adult male small RNAs matching pre-miRNA hairpins in the sense (blue) or antisense (red) orientation.

Fig 4. Size distribution and sequence logos for transcriptome-matching small RNAs in adult males (first part: Libraries #1 and 2).

See Supplementary S1 File, section 3, for the other developmental stages. A: Library #1, B: Library #2. Numbers of reads are expressed as parts per million (ppm) after normalization to the total number of genome-matching reads that do not match abundant non-coding RNAs. For each orientation (sense or antisense-transcriptome-matching reads), a logo analysis was performed on each size class (18 to 30 nt long RNAs).

Fig 5. Size distribution and sequence logos for transcriptome-matching small RNAs in adult males (second part: Libraries #3 and 4).

See Supplementary S1 File, section 3, for the other developmental stages. A: Library #3, B: Library #4. Numbers of reads are expressed as parts per million (ppm) after normalization to the total number of genome-matching reads that do not match abundant non-coding RNAs. For each orientation (sense or antisense-transcriptome-matching reads), a logo analysis was performed on each size class (18 to 30 nt long RNAs).