Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8

Abstract

Background Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. Results We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. Conclusion Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

In depth exploration of the alternative proteome of Drosophila melanogaster

“…With more than 300 AltProts produced from uORFs, intORFs or dORFs, our data strongly advocate towards a polycistronic nature of mRNA in Drosophila melanogaster (Figure 2). Of note, 52 AltProts are produced from previously predicted long non-coding RNA (Figure 2), including 1 AltProt encoded by a precursor of miRNA (pri-miRNA) (Figure 2), supporting the idea that miPEPs (miRNA-encoded peptides) are expressed in flies (Immarigeon et al, 2021;Montigny et al, 2021). Regarding the sources of production of AltProts, and more particularly the chromosomes they are produced from, a distribution similar to the predicted AltProts distribution from OpenProt was observed (Supplementary Figure 2A-B), although slight differences could be noticed.…”

“…Contrasting with these results, we did not find many unannotated proteins with a coding sequence of more than 100 codons, revealing that the annotation of proteins with ORF of 100 codons or more is precise and reliable. On the other hand, our study shows that many proteins of less than 100 amino acids remain to be discovered, especially considering the fact that we did not search for alternative proteins of less than 30 amino acids, which are known to be expressed and functional in D. melanogaster (Magny et al, 2013; Zanet et al, 2015; Immarigeon et al, 2021; Montigny et al, 2021) and would require further investigation. Interestingly, these AltProts are not necessarily produced from the first predicted ORF on an RNA (Figure 3), one spectacular result came from an AltProt being synthesized from the 134 th predicted ORF on the dumpy mRNA (Supplementary table 1).…”

“…D. melanogaster adult flies and embryos were maintained and collected as previously described (Fabre et al, 2019). S2 cells were cultured as described in (Montigny et al, 2021).…”

“…For imaging experiments, S2 cells were co-transfected using actin-GAL4 driver with UAS-CG34150-GFP and UAS-AltProtCG34150-RFP or UAS-CG2650-GFP and UAS-AltProtCG2650-RFP (both constructions encoding AltProts also contain the start codons and sequence of the canonical proteins). Cells were transfected with effectene (Qiagen) according to manufacturer specification and as described in (Montigny et al, 2021). 48h post transfection, cells were fixed in 4% formaldehyde in phosphate buffer saline (PBS) at room temperature for 30 minutes.…”

From Yeast to Mammals, the Nonsense-Mediated mRNA Decay as a Master Regulator of Long Non-Coding RNAs Functional Trajectory

“…Numerous methods have been developed to assess this possibility [148][149][150]. Additionally, a growing body of experimental data indicate that "non-coding" RNAs can indeed be translated [151], including not only lncRNAs but also circular RNAs [152][153][154] and primary microRNAs transcripts [155,156]; moreover, these translation events can produce functional peptides [134,151,[157][158][159].…”