Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

Abstract

Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

Fig 1. The length distribution of small RNA reads of E. huxleyi.

Fig 2. Distribution of unique smRNA reads with lengths between 18 and 26 nt in the smRNA library.

Fig 3. Expression of miRNA Precursors in calcifying and non-calcifying E. huxleyi cells grown in filtered seawater media.

Expression values are relative to that of the, U6 small nuclear RNA reference gene and were determined using the 2^Δct method.

Fig 4. Differential expressions of miRNA in calcifying versus non-calcifying cells.

Cultures were grown in filtered seawater media, expression normalized to the U6 small nuclear RNA, and variance determined by the 2^ΔΔCT method.

Fig 5. The mapping of small RNAs relative to the annotation of E. huxleyi genome.

If a read is mapped to more than one feature other than intergenic region, the most frequent one is selected.

Fig 6. Domain architecture variation in DICER homologs from E. huxleyi and other simple eukaryotes.

The RNase III domain (pink) is responsible for cleaving dsRNA while the helicase domain (purple) is important for unwinding of the dsRNA, the DSRM domain (blue) binds double stranded RNA, and the PAZ domain (yellow) binds to the 3’ end of the target ds RNA. The more complex enzymes in higher eukaryotes typically contain all four domains whereas the ancestral DICERs from unicellular eukaryotes are generally missing one or more domains.

Fig 7. A comparison of argonaute PAZ and PIWI domains from E. huxleyi, Arabidopsis, Chlamydomonas, Homo sapiens, Drosophila, and Nannochloropsis.

Several residues in the PAZ domain (A) marked by astericks, are invariant in the E. huxleyi argonaute (226029) and thought to stabilize the dsRNA-binding region. These include: 1) a subdomain of aromatic residues, 2) a cysteine residue preceded by a proline and a glutamine, and 3) other conserved residues that form a hydrophobic subdomain that interacts with RNA (Firmino et al., 2005). Amino acids in the core motifs of the PIWI domain (B) are also well conserved across the E. huxleyi homologs, and the catalytic DDH triad marked by asterisks is invariant. Coloring is based on the degree of amino acid homology.