Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

Abstract

Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3ʹ-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.

Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity

“…Thus far, D. discoideum is the only Amoebozoan in which miRNAs have been convincingly identified [30][31][32][33] . The D. discoideum miRNAs are mostly expressed from single intergenic regions and are upregulated upon multicellular development.…”

Fractional 2′-O-methylation in the ribosomal RNA of Dictyostelium discoideum supports ribosome heterogeneity in Amoebozoa

“…Using these settings, we identified 577 box C/D snoRNA candidates in the genome of D. discoideum (data not shown), including the set described before 43 . To refine our search, we next addressed the expression of these candidates in publicly available RNA-seq data of the axenic AX2 wild type strain, deposited in duplicate 47 at the sequence read archive ( https://www.ncbi.nlm.nih.gov/sra ). Specifically, we mapped reads to the genomic loci of the candidates and selected only those sequences that exceeded a read count of 100 and were not part of a longer transcript, as indicated by a distinct 5′ end.…”

“…Furthermore, we could not obtain a product for several snoRNAs, despite the use of several distinct primers in these experiments. Therefore, to obtain a more complete view on box C/D snoRNA accumulation, we retrieved RNA-seq datasets for AX2 and ∆drnB in axenic growth and in the slug stage of development from NCBI, which were originally deposited by Liao et al 47 . As a first step, we performed a principal component analysis (PCA) of box C/D snoRNA expression on two biological replicates for each time point per strain.…”

“…Accession numbers of the utilized data sets can be found in Supplementary Table S1 . Sample preparation and sequencing was described in Liao et al 47 .…”

Dicer structure and function: conserved and evolving features

“…There are other Dicers, whose architecture deviates even more. For example, DrnA/B in Dictyostelium retains the tandem RNase III core module, the dsRBD is placed at the N terminus and the central part of the protein does not show sequence homology with canonically built Dicers (Hinas et al , 2007; Kruse et al , 2016; Liao et al , 2018; Fig 1A). How DrnA/B determine the length of the product remains unclear.…”