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.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate

“…3b), we questioned whether post-transcriptional regulation by microRNAs could be involved. We found expected components of RNAi machinery in A. gibbosum consistent with previous reports [7,[42][43][44][45] (Fig. 3c and Additional file 1: Supplementary Fig.…”

Current Knowledge and Recent Advances in Marine Dinoflagellate Transcriptomic Research

“…However, not all protein coding genes are regulated by miRNAs. Dagenais-Bellefeuille et al [35] recently found that circadian regulation gene expression of luciferase binding protein in Lingulodinium polyedrum is not mediated by miRNAs, although transcriptomics data show the presence of miRNAs in L. polyedrum.…”

Translation and Translational Control in Dinoflagellates

“…This suggests that these organisms must have an alternative to Dicer in order to generate their small RNAs. BLAST searches have identified Dicer-like proteins in Perkinsus marinus , a basal lineage in the dinoflagellate clade, as well as in transcriptomes from the core dinoflagellates Symbiodinium and Lingulodinium [ 168 ]. This indicates that Dicer-like enzymes and Argonaute-like proteins are present, although RdRp related proteins have not yet been detected.…”

“…The fact that miRNAs can in some cases mediate translation rates without affecting mRNA levels is potentially applicable to understanding circadian regulation of translation in Lingulodinium where changes in protein synthesis rates are not accompanied by changes in RNA levels. This was tested explicitly using small RNA sequencing to detect miRNAs in this species [ 168 ]. However, the results indicate miRNAs do not regulate circadian translation, as out of 9787 sequence reads corresponding to LBP only six were found in an antisense orientation, and none of the 5749 reads corresponding to luciferase were in an antisense orientation.…”