Small ORF-encoded peptides: exploring a novel repertoire of regulators for Drosophila morphogenesis – MorphoSmORF

Over the past decade, high throughput sequencing has revealed the existence of a myriad of long RNAs molecules, generally referred to as long non-coding RNAs (lncRNAs) since they lack large (i.e. > 100 codons) protein-coding ORF. However, genome-wide ribosome profiling now demonstrates that lncRNAs are often translated and produce a large diversity of peptides encoded from small ORF (smORF). LncRNA thus emerge as an overlooked source of uncharacterized peptides, and case studies from yeast, to plants and animals show that smORF peptides play key developmental functions. This novel “micro-proteome” therefore shifts our view of the coding genome and opens several questions concerning the functional repertoire and general significance of smORF peptides. A major challenge now resides in elucidating the putative functions and mode of action of smORF peptides.
Building on our expertise and accumulated data, the MorphoSmORF proposal aims at using functional approaches in Drosophila to decipher the molecular activity of Pri smORF peptides in vivo and to identify novel regulatory smORF peptides in the control of morphogenesis. Our recent outcomes have demonstrated the key role of Pri smORF peptides (11 to 32 amino-acids length) in epidermis development and identified their molecular target. Pri smORF peptides are essential to embryonic and post-embryonic development, their inactivation leading to severe defects in epidermal differentiation and morphogenesis of adult legs. We showed that Pri peptides act to control the activity of the transcription factor Shavenbaby (Svb), a well-known key regulator of epidermal development. From genome-wide functional screening, we further identified an E3 ubiquitin ligase, Ubr3, as the direct target of Pri peptides. Upon binding to Ubr3, Pri peptides allow the binding of Ubr3 to the Svb N-terminus and subsequent ubiquitination of Svb on three highly conserved lysine residues. Once ubiquitinated, Svb is targeted to the proteasome where it undergoes a limited degradation, which removes a repressor domain thereby switching Svb activity towards a transcriptional activator. Ultimately, this processed form of Svb then activates the expression of a large battery of effector target genes, collectively responsible for epidermal differentiation. How Pri peptides modify the properties Ubr3 remains to be fully elucidated, as well as which are the functional partners involved in this regulatory mechanism, and how are they redeployed across development, in particular to explain their role in appendage morphogenesis.
The specific objectives of MorphoSmORF are:
1) elucidating the molecular mode of action of Pri peptides on the regulation of proteasome activity and identifying key functional partners;
2) deciphering the function of Pri peptides for adult leg morphogenesis and identifying the molecular targets of Pri peptides in this tissue.
3) identifying novel smORF peptides regulating leg development in flies, combining bioinformatic prediction, in vivo functional profiling, and biochemical analysis of their molecular activity and targets.
Based on our accumulated expertise and preliminary data, the MorphoSmORF proposal incorporates a complementary array of approaches (biochemistry, transcriptional regulation, live imaging, bio-informatics and CRISPR/Cas9 method) and is supported by a dense network of French and international collaborations with experts in respective fields (proteasome regulation, leg patterning and tissue remodeling, genome editing and bio-informatics). The expected outcomes of MorphoSmORF will provide significant advances in our basic knowledge on the emerging field of smORF peptides, of direct relevance for biomedical and agronomical research.