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Feeding an increasing world population in the context of climate change is one of the grand challenges faced by our generation. Here, the authors show that external application of synthetic complementary peptides can increase the abundance of target proteins to modulate plant growth or stress resistance.

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.

Summary: MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

In vertebrates, skeletal myogenesis involves the sequential activation of myogenic factors to lead ultimately to the differentiation into slow and fast muscle fibers. How transcriptional co-regulators such as arginine methyltransferases PRMT4/CARM1 and PRMT5 control myogenesis in vivo remains poorly understood. Loss-of-function experiments using morpholinos against PRMT4/CARM1 and PRMT5 combined with in situ hybridization, quantitative polymerase chain reaction, as well as immunohistochemistry indicate a positive, but differential, role of these enzymes during myogenesis in vivo. While PRMT5 regulates myod, myf5 and myogenin expression and thereby slow and fast fiber formation, PRMT4/CARM1 regulates myogenin expression, fast fiber formation and does not affect slow fiber formation. However, our results show that PRMT4/CARM1 is required for proper slow myosin heavy chain localization. Altogether, our results reveal a combinatorial role of PRMT4/CARM1 and PRMT5 for proper myogenesis in zebrafish.

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.