No description available.

Small open reading frame genes (smORFs) are a new class of genes, which emerged from the revision of the idea that open reading frames have to be longer than 100 codons to be protein coding and functional. Although bio-informatics evidence suggests that thousands of smORF genes could exist in any given genome, proof of their functional relevance can only be obtained through their functional characterization. This work represents such a study for a Drosophila smORF (pncr003;2L), which was initially misannotated as a non-coding RNA because of its lack of a canonical long open reading frame. Here I show that pncr003;2L codes for two small peptides of 28 and 29 aa, expressed in somatic and cardiac muscles. After generating a null condition for this gene, I use the adult Drosophila heart as a system to assess the function of pncr003;2L. With this system, I show that the small pncr003;2L peptides regulate heart contractions by modulating Ca2+ cycling in cardiac muscles, with either lack or excess of function of these peptides leading to cardiac arrhythmias, and abnormal calcium dynamics. Finally, through an extensive homology study, I show that these small peptides share a great amount of structural and functional homology with the peptides encoded by the vertebrate smORFs sarcolipin (sln) and phospoholamban (pln), which act as major regulators of the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), the channel responsible for calcium uptake into the ER following muscle contraction. These results highlight the importance of the pncr003;2L smORF and the Drosophila system, for the study of cardiac pathologies, but most importantly, they show that this family of peptides, conserved across evolution, represent an ancient system for the regulation of calcium trafficking in muscles. This work corroborates the prevalence, and relevance of this novel class of genes, and shows that closer attention should be given to smORFs in order to determine the full extent of their biological contribution.

During the formation of the insect and mammalian nervous system the embryo activates specific programs of cellular differentiation along the main body axis so that the specification and organization of neural cells is set in coordination with axial level. At the genetic level such cellular specification programs rely on the regulated expression of a family of transcription factors encoded by the Hox genes. However, the precise molecular mechanisms controlling Hox expression in the nervous system are not well understood. In this thesis we investigate the molecular mechanisms underlying Hox gene expression within the Drosophila central nervous system (CNS) with a focus on post-transcriptional control via RNA binding proteins (RBP) and microRNAs (miRNAs). Much of the work is centred on the analysis of the Hox gene Ultrabithorax (Ubx) as this is the Hox gene for which post-transcriptional regulation is currently best understood. Through the combination of genetic, molecular and imaging methods we first show that the pan-neural RBP ELAV regulates Ubx RNA processing and protein expression during the embryonic development of the CNS. Secondly, using a suite of genetic and behavioural methods we report that Ubx repression by miRNAs encoded within the iab-4/iab-8 locus (miR-iab4/iab8) is required for the coordination of a specific larval behaviour: self-righting behaviour. Third, we explore the cellular basis of larval self-righting behaviour in the context of miRNA-dependent Ubx regulation and find that: (i) removal of miR-iab4/iab8 does not lead to major anatomical defects in the CNS or muscles; (ii) artificial increase in UBX protein expression in cholinergic interneurons disrupts self-righting behaviour; and (iii) UBX protein expression in cholinergic interneurons is regulated by miR-iab4/iab8. These observations imply that UBX regulation by miR-iab4/iab8 in cholinergic interneurons controls self-righting behaviour. Altogether our work adds to the current understanding of the molecular mechanisms underlying Hox gene expression during CNS formation and gives new insights on the role of RBP and miRNA regulation on the control of gene expression and behaviour.

The Hox genes encode a family of transcriptional regulators that activate distinct developmental programs along the anterior-posterior (AP) axis of animals. Recent observations in Drosophila demonstrate that at least two miRNAs can repress Hox gene expression during development suggesting that miRNA-based regulation might be a general mechanism of Hox gene regulation. Here explore this possibility by applying a comprehensive genetic approach to identify miRNAs able to repress Hox gene expression during development. Given that the reduction of Drosophila Hox gene Ultrabithorax (Ubx) expression leads to easily tractable homeotic transformations in haltere, I use Ubx to test the repressive effects of dozens of miRNAs in an overexpression screen. Scoring over 10,000 halteres showed that out of 106 miRNAs tested, ~28% produced Ubx mutant phenotypes suggesting that miRNA-dependent Hox regulation might be a pervasive mechanism controlling Hox gene function during development. I classify phenotypes into four major categories: Ubx mutant effects (Class I and II) and others (Class III and IV). Through the combination of RNA-Seq data and TaqMan RT-PCR approaches, I confirm that there is no correlation between the phenotypic strength and miRNA expression level indicating that haltere phenotypes emerge from miRNA qualitative roles. Furthermore, using protein expression analysis and Ubx 3' UTR fluorescent reporters, I confirmed that at least nine miRNAs affect Ubx protein expression and that six of these directly target Ubx 3' UTR in vivo. Lastly, I explore the nature and effects of miRNA regulation of Ubx at the cellular level in the Drosophila embryonic CNS and find that miR-252 is sufficient and necessary to repress Ubx expression in specific neural lineages. Our work thus contributes to the understanding of miRNA-mediated Hox gene regulation and, more generally, to the study of miRNA-target interactions within the physiological context of metazoan development.