The microRNA Pathway of Macroalgae: Its Similarities and Differences to the Plant and Animal microRNA Pathways

Abstract

In plants and animals, the microRNA (miRNA) class of small regulatory RNA plays an essential role in controlling gene expression in all aspects of development, to respond to environmental stress, or to defend against pathogen attack. This well-established master regulatory role for miRNAs has led to each protein-mediated step of both the plant and animal miRNA pathways being thoroughly characterized. Furthermore, this degree of characterization has led to the development of a suite of miRNA-based technologies for gene expression manipulation for fundamental research or for use in industrial or medical applications. In direct contrast, molecular research on the miRNA pathway of macroalgae, specifically seaweeds (marine macroalgae), remains in its infancy. However, the molecular research conducted to date on the seaweed miRNA pathway has shown that it shares functional features specific to either the plant or animal miRNA pathway. In addition, of the small number of seaweed species where miRNA data is available, little sequence conservation of individual miRNAs exists. These preliminary findings show the pressing need for substantive research into the seaweed miRNA pathway to advance our current understanding of this essential gene expression regulatory process. Such research will also generate the knowledge required to develop novel miRNA-based technologies for use in seaweeds. In this review, we compare and contrast the seaweed miRNA pathway to those well-characterized pathways of plants and animals and outline the low degree of miRNA sequence conservation across the polyphyletic group known as the seaweeds.

Keywords: animal miRNA pathway; miRNA conservation; miRNA evolution; miRNA pathway; microRNA (miRNA); plant miRNA pathway; seaweed miRNA pathway.

Figure 1

The miRNA pathway of the model plant Arabidopsis thaliana. In the nucleus of the Arabidopsis cell, Pol II transcribes the pri-miRNA from a MIR gene. The pri-miRNA is bound by SE1 and is transported to the D-body where it is processed by DCL1/DRB1 into a pre-miRNA and then a miRNA/miRNA* duplex. HEN1 methylates the 3′ terminus of both duplex strands and AGO1 retains the miRNA guide strand and discards the miRNA* passenger strand. HST is involved in the nucleus-to-cytoplasm export of some miRNAs, and in the cytoplasm of the Arabidopsis cell, AGO1 uses its loaded miRNA to guide the silencing of target gene transcripts predominantly via mRNA cleavage but also via translational repression. HESO1 controls the steady-state levels of Arabidopsis miRNAs to add an additional layer of complexity to miRNA-directed gene expression regulation. AGO1, ARGONAUTE1; D-body, nuclear Dicing body; DCL1, DICER-LIKE1; DRB1, dsRNA BINDING1; HEN1, HUA ENHANCER1; HESO1, HEN1 SUPPRESSOR1; MIR, MICRORNA gene; Pol II, RNA polymerase II; pro, MIR gene promoter; SE1, SERRATE1; ter, MIR gene terminator.

Figure 2

The miRNA pathway of the model animal Drosophila melanogaster. In the nucleus of the Drosophila cell, Pol II transcribes the pri-miRNA from a MIR gene. Pol II is also responsible for the transcription of mirtrons from host genes or for the transcription of MIR gene clusters. After folding into a stem-loop dsRNA structure, the pri-miRNA is bound by Ars2 and is then processed into a pre-miRNA by Drosha/Pasha. The pre-miRNA is exported from the nucleus to the cytoplasm by the RanGTP-dependent exportin, XPO5. In the cytoplasm of the Drosophila cell, the pre-miRNA is further processed into the miRNA/miRNA* duplex by Dcr1/Loqs. After duplex strand separation and degradation of the miRNA* passenger strand, Ago1 uses the loaded miRNA to guide the silencing of target gene transcripts via translational repression, a mode of miRNA-directed gene expression regulation that also requires GW182. Ago1, Argonaute1; Ars2, Arsenite resistance protein2; Dcr1, Dicer1; GW182, glycine/tryptophan repeat protein 182; Loqs, Loquacious; MIR, microRNA gene; mirtron, miRNA precursor-containing intron; Pol II, RNA polymerase II; pro, MIR gene promoter; Ran-GTP, RAS-related nuclear protein GTPase; ter, MIR gene terminator; XPO5, Exportin-5.

Figure 3

The miRNA pathway of the model green microalgae Chlamydomonas reinhardtii. In the nucleus of a Chlamydomonas cell, Pol II transcribes pri-miRNAs directly from MIR genes, or from protein-coding host genes with the mirtron pri-miRNA forming post intron splicing. Post folding into a stem-loop dsRNA structure, the pri-miRNA is processed into a pre-miRNA by the DCL3/DUS16 partnership. DCL3/DUS16 are also required for further processing of the pre-miRNA into the miRNA/miRNA* duplex to indicate that all processing steps of the production stage of the Chlamydomonas miRNA pathway occur in the nucleus. The protein mediator of the export of the miRNA/miRNA* duplex out of the nucleus and into the cytoplasm of a Chlamydomonas cell remains unknown (red boxed question mark). In the cytoplasm, the miRNA/miRNA* duplex strands are separated from each other by an unknown mechanism, and then the miRNA* passenger strand is degraded. AGO3 retains the miRNA guide strand and uses it to direct expression regulation of target gene transcripts via either a mRNA cleavage or translational repression mode of RNA silencing. The steady state levels of Chlamydomonas miRNAs are controlled by MUT68, together with RRP6, to add an additional layer of regulatory complexity to miRNA-directed gene expression control in Chlamydomonas. AGO3, Argonaute3; DCL3, Dicer-like3; DUS16, Dull slicer-16; MIR, MICRORNA gene; mirtron, miRNA precursor containing intron; MUT68, mutant 68 (terminal nucleotidyltransferase); Pol II, RNA polymerase II; pro, MIR gene promoter; RRP6, Ribosomal RNA processing6; ter, MIR gene terminator; ?, unknown protein factor.

Figure 4

Proposed miRNA pathway for the red seaweed Asparagopsis taxiformis. (A) Pol II transcribes pri-miRNAs from MIR genes or mirtron precursors from protein-coding host genes. DCL3, with the assistance of the RNA-binding protein SE1, processes pri-miRNAs into pre-miRNAs and then pre-miRNAs into miRNA/miRNA* duplexes in the nucleus of A. taxiformis cells. The miRNA/miRNA* duplex is exported from the nucleus to the cytoplasm of the A. taxiformis cell by a Ran-GTP-dependent XPO5-mediated process and is loaded into AGO3. After the removal and degradation of the miRNA* passenger strand, AGO3 uses the loaded miRNA as a sequence specificity guide to direct the silencing of miRNA target genes via either mRNA cleavage or the translational repression mode of gene expression control. The steady-state levels of miRNAs are controlled in the A. taxiformis cell cytoplasm by the activity of the nucleotidyltransferase, HESO1. AGO3, ARGONAUTE3; DCL3, DICER-LIKE3; HESO1, HEN1 SUPPRESSOR1; MIR, MICRORNA gene; mirtron, miRNA precursor-containing intron; Pol II, RNA polymerase II; pro, MIR gene promoter; ter, MIR gene terminator; Ran-GTP, RAS-related nuclear protein GTPase; SE1, SERRATE1; XPO5, EXPORTIN-5. (B) Schematic representation of the functional domain landscape of Asparagopsis homologs of the core machinery proteins of the Arabidopsis, Drosophila, and Chlamydomonas miRNA pathways including Ata-DCL3, Ata-SE1, Ata-XPO5, Ata-RanGTP, Ata-AGO3, and Ata-HESO1. Ago-NTD, Argonaute N-terminal domain; ALD, Argonaute linker domain; ARM, Armadillo repeat domain; Ars2, Arsenite-resistance2 domain; DExD/Hel, DExD/H-box helicase domain; DUF4187, domain of unknown function 4187; dsRBD, dsRNA-binding domain; INT, Importin-β N-terminal domain; PAP, nucleotide poly A polymerase domain; PAZ, Piwi, Argonaute, and Zwille domain; PIWI, Piwi domain; Ran-GTP, Ran small GTPase domain; RNase III, Ribonuclease III domain; SE1, Serrate1/Ars2 N-terminal domain; TUTase, nucleotidyltransferase domain; XPO1, Exportin1-like domain.

Figure 5

Proposed best practice approach to miRNA identification, characterization, and technology development in seaweeds. The flowchart outlines the four key steps for identifying, analyzing and validating miRNAs in seaweeds. The process is divided into four major modules, which include (1) sample collection, (2) identification of core protein machinery and the inference of miRNA-directed regulatory mechanisms, (3) sRNA sequencing for sRNA library generation and miRNA identification and annotation via bioinformatics, together with experimental validation, and (4) application development. Quality control measures are emphasized at each step to ensure reliability and accuracy in miRNA discovery and functional validation.