miRNAs Do Not Regulate Circadian Protein Synthesis in the Dinoflagellate Lingulodinium polyedrum

Abstract

Dinoflagellates have been shown to express miRNA by bioinformatics and RNA blot (Northern) analyses. However, it is not yet known if miRNAs are able to alter gene expression in this class of organisms. We have assessed the possibility that miRNA may mediate circadian regulation of gene expression in the dinoflagellate Lingulodinium polyedrum using the Luciferin Binding Protein (LBP) as a specific example. LBP is a good candidate for regulation by miRNA since mRNA levels are constant over the daily cycle while protein synthesis is restricted by the circadian clock to a period of several hours at the start of the night phase. The transcriptome contains a potential DICER and an ARGONAUTE, suggesting the machinery for generating miRNAs is present. Furthermore, a probe directed against an abundant Symbiodinium miRNA cross reacts on Northern blots. However, L. polyedrum has no small RNAs detectable by ethidium bromide staining, even though higher plant miRNAs run in parallel are readily observed. Illumina sequencing of small RNAs showed that the majority of reads did not have a match in the L. polyedrum transcriptome, and those that did were almost all sense strand mRNA fragments. A direct search for 18-26 nucleotide long RNAs capable of forming duplexes with a 2 base 3' overhang detected 53 different potential miRNAs, none of which was able to target any of the known circadian regulated genes. Lastly, a microscopy-based test to assess synthesis of the naturally fluorescent LBP in single cells showed that neither double-stranded nor antisense lbp RNA introduced into cells by microparticle bombardment prior to the time of LBP synthesis were able to reduce the amount of LBP produced. Taken together, our results indicate that circadian control of protein synthesis in L. polyedrum is not mediated by miRNAs.

Fig 1. Gel electrophoretic analysis of miRNAs.

(A) A 22 base miRNA highly expressed in S. kawagutii was tested by Northern blots on RNA extracted from the higher plant Arabidopsis thaliana and the dinoflagellates S. kawagutii and L. polyedrum. RNA samples were electrophoresed on acrylamide gels with ssDNA oligonucleotides of 17, 21 and 25 bases as size markers. (B) RNA extracted from the higher plant Solanum chacoense contains small RNA of 22 bases in length, whereas no RNA of this size class is detected in samples from the dinoflagellates L. polyedrum, Amphidinium carterae or Pyrocystis lunula.

Fig 2. Schematic Dicer domain structures.

The domain structures of animal, plant, diatom and dinoflagellate DICERs as determined by the conserved domain function in the blastp package.

Fig 3. Patterns of read mapping to four different reference sequences.

Twenty-eight Trinity sequences, corresponding to the 100 most abundant reads in the 4.9 M filtered sequence dataset, showed three patterns of assembled reads. The first (16 sequences) had reads lying all in the same direction (A). When the direction of the Trinity sequence encoding a protein was known, these reads correspond to the sense orientation. A second pattern contains very few sequences in the antisense direction, and these usually differ in sequence at their ends (B). A third pattern (C) had numerous antisense sequences, but these are distributed over the length of the transcript. The reference sequence in this case was a mitochondrial gene (cox1). All examples are all drawn to scale on the horizontal axis. The vertical axis shows the coverage (number of reads) at each base. Dark green indicates reads lie in both directions, while light green indicates all reads have the same orientation.

Fig 4. Patterns of read mapping to LBP, GAPCp or Rubisco as a reference sequence.

(A) The majority of the reads mapped to LBP from the 4.9 million read dataset correspond to the sense strand. Six antisense sequences were found (dark green), and these potentially form duplexes with 3’ overhangs with six of the sense orientation reads as shown. (B) GAPCp. (C) Rubisco.

Fig 5. Sequence logo of sequences similar to S. kawagutii miRNAs.

All but 27 reads from 25,195 with similarity to the 101 S. kawagutii miRNA could be aligned. Identical residues in the sequence logo correspond to the sequence of the probe used on Northern blots (5’-ACAGATTGCGGCAACCGTGCAG-3’).

Fig 6. Length distribution of potential L. polyedrum miRNAs.

The majority of the 53 unique small RNAs among the 94,239 collapsed sequences that are able to form duplexes with a 2 base 3’ overhang are 22 bases long.

Fig 7. Fluorescence and bright field micrographs of L. polyedrum.

A three dimensional reconstruction of a night phase (A-C) and a day phase (D-F) cell from a stack of confocal images shows scintillon fluorescence (A, D; Ex: 405 nm, Em: 420–480 nm), or for chlorophyll fluorescence (B, E; Ex: 405 nm, Em >575 nm) and a merged view of the two (C, F). The difference in scintillons numbers is under circadian control. Samples of cell cultures bombarded with tungsten microparticles show that small (~ 2 μm) microparticle aggregates (G, J) can be visualized inside cells (black arrows) by differential interference contrast (DIC) microscopy. The same fields examined for scintillon fluorescence (H, K) illustrates that cells with microparticles can have normal numbers of scintillons (H) or show the dark cell phenotype (K; white arrowheads); cells without microparticles can also show the dark phenotype (K). Chlorophyll fluorescence (I, L) in plastids serves as a marker for cell integrity. Scale bars are 10 μm.

Fig 8. LBP RNAi does not significantly alter the luciferin fluorescence in cells.

A) Cells with microparticles (arrow heads) under higher magnification (scale bar is 10 um). B) Total luciferin fluorescence in cells bombarded with microparticules lacking RNA at ZT12 and observed at ZT18, as well as cells bombarded with microparticules charged with lbp RNA at ZT12 and observed at ZT18 either on the same day (ZT18-1) or the following day (ZT18-2).