Drosophila Regnase-1 RNase is required for mRNA and miRNA profile remodelling during larva-to-adult metamorphosis

Abstract

Metamorphosis is an intricate developmental process in which large-scale remodelling of mRNA and microRNA (miRNA) profiles leads to orchestrated tissue remodelling and organogenesis. Whether, which, and how, ribonucleases (RNases) are involved in the RNA profile remodelling during metamorphosis remain unknown. Human Regnase-1 (also known as MCPIP1 and Zc3h12a) RNase remodels RNA profile by cleaving specific RNAs and is a crucial modulator of immune-inflammatory and cellular defence. Here, we studied Drosophila CG10889, which we named Drosophila Regnase-1, an ortholog of human Regnase-1. The larva-to-adult metamorphosis in Drosophila includes two major transitions, larva-to-pupa and pupa-to-adult. regnase-1 knockout flies developed until the pupa stage but could not complete pupa-to-adult transition, dying in puparium case. Regnase-1 RNase activity is required for completion of pupa-to-adult transition as transgenic expression of wild-type Drosophila Regnase-1, but not the RNase catalytic-dead mutants, rescued the pupa-to-adult transition in regnase-1 knockout. High-throughput RNA sequencing revealed that regnase-1 knockout flies fail to remodel mRNA and miRNA profiles during the larva-to-pupa transition. Thus, we uncovered the roles of Drosophila Regnase-1 in the larva-to-adult metamorphosis and large-scale remodelling of mRNA and miRNA profiles during this metamorphosis process.

Keywords: RNase; Regnase-1; drosophila; mRNA; metamorphosis; miRNA.

Figure 1.

RNase catalytic activity of Regnase-1 is required for the larva-to-adult metamorphosis. (a) Domain structures of Drosophila Regnase-1 (CG10889) and human Regnase-1. Amino acid sequence alignment of Regnase-1 orthologues around the residues mutated in Figure 1(e) is also shown. Dme, Drosophila melanogaster; Cel, Caenorhabditis elegans; Dre, Danio rerio; Hsa, Homo sapiens. (b) Knockout (KO) allele of Drosophila Regnase-1 created in this study. Nucleotide residues targeted by sgRNA is italicised. The 5-nt residues deleted are shown in green. Amino acid residues after the frameshift mutation in the KO allele are shown in magenta. (c) Life cycle of Drosophila. (d) mRNA expression levels of regnase-1, drosha, dicer-1, and RpL32, in wild-type third instar larvae and early pupae, determined by mRNA-seq. Mean ± SD for three biological replicates. (e) Representative images of third instar larvae, early pupae, dead late pupae, and eclosed adult flies, for indicated genotypes. Successful metamorphosis rates are shown with the numbers of successful metamorphosis event/the numbers of examined pupae shown in parenthesis.

Figure 2.

miRNA profile is dysregulated in regnase-1 knockout early pupae. Heatmaps of miRNA and endo-siRNA expression levels determined by high-throughput sequencing of small RNAs. Means of three biological replicates were used. (a-c) show the same data using different normalisation references and in different orders of miRNAs. (a) miRNA and siRNA abundance relative to those in wild-type (+/+) third instar larvae. miRNAs are sorted in descending order of the fold-changes in regnase-1^-/- (-/-) third instar larvae. (b) miRNA and siRNA abundance relative to those in wild-type (+/+) early pupae. miRNAs are sorted in descending order of the fold-changes in regnase-1^-/- (-/-) early pupae. (c) miRNA and siRNA abundance relative to those in wild-type (+/+) third instar larvae. miRNAs are sorted in descending order of the fold-changes in wild-type early pupae. Only miRNAs whose mean abundance was more than 100 reads per million total reads in either regnase-1+/+, -/+ or -/- samples and three endo-siRNAs (esi-1.1, esi-1.2, and esi-2.1) are shown. Third instar larva is shown as ‘larva’ for simplicity.

Figure 3.

Regnase-1 is required for remodelling miRNA profile during the larva-to-pupa transition. (a-b) Abundance of top five miRNAs that are significantly (a) upregulated and (b) downregulated in regnase-1^-/- early pupae compared with control early pupae, determined by high-throughput sequencing. Mean ± SD for three biological replicates. P-value <0.05 is indicated by *. Third instar larvae are shown as ‘Larvae’ for simplicity.

Figure 4.

Regnase-1 is required for normal mRNA profile in pupae. (a) Sample-to-sample distance matrix of mRNA profiles determined by mRNA-seq. (b) Principal component analysis of mRNA profiles. Third instar larvae are shown as ‘larvae’ for simplicity.

Figure 5.

MA plots show that Regnase-1 is required for normal mRNA profile in pupae. MA plots of mRNA expression. x-axis shows the means of normalised counts among the six biological samples tested (three biological replicates each for two genotypes or developmental stages that are compared in each graph). y-axis shows log2 fold-change in the normalised counts between the two genotypes or developmental stages. (a) MA plots of mRNA profiles in third instar larvae, comparing pair-wisely among regnase-1 +/+, -/+, and -/-. (b) MA plots of mRNA profiles in early pupae, comparing pair-wisely among regnase-1 +/+, -/+, and -/-. (c) MA plots of mRNA profiles comparing between early pupae and third instar larvae of regnase-1+/+, -/+, and -/-. Third instar larvae are shown as ‘Larvae’ for simplicity.

Figure 6.

Regnase-1 is required for remodelling mRNA profile during the larva-to-pupa transition. Abundance of top five mRNAs that are significantly (A, B) upregulated and (C, D) downregulated in regnase-1^-/- early pupae compared with control early pupae, determined by high-throughput sequencing. (A,C) Normalised counts in RNA-seq. (B,D) Relative expression normalized by rp49 determined by qRT-PCR. Expression relative to wild-type (regnase-1^+/+) third instar larvae are shown except in fln, where expression relative to wild-type (regnase-1^+/+) early pupae larvae are shown. Mean ± SD for three biological replicates. P-value <0.05 is indicated by *. Third instar larvae are shown as ‘Larvae’ for simplicity.