In vivo analysis of the coupling between alternative splicing poly-adenylation and miRNA regulation in the Drosophila Hox gene Ultrabithorax

The instructions to build an animal are all encoded in its compact genetic material. This is found in the cell nuclei, in the form of discrete DNA sequences defined as genes. Genes are copied into RNA messages, which will survive for variable times within the cell and finally be translated into proteins. It is the proteins, in the right amount and with their particular enzymatic or structural properties that will actually define the shape and functions of animal tissues and organs. We could say that genes provide the plans and ideas, and proteins make them happen. Since all cells in a given animal have exactly the same genes, an important question in modern biology is to determine 'how, when and where' RNA messages are 'expressed' enabling their final products to control particular aspects of animal formation, so that for example, legs end up being different from eyes and brains. To study this question, most biologists, have concentrated their research on how genes are switched 'ON'. This is how a DNA sequence is copied into an RNA message. However, once made RNA messages can suffer alterations in their structure (splicing) that may make them survive for a long time or be destroyed very quickly. My work is focused precisely on the mechanisms that are able to alter RNA structures so that they may be degraded faster or more slowly. Think that if the RNA is eaten away it will not produce any protein, and the gene will not achieve any function for the cell or the organism. In this project I will study a particular gene, one from the fruitfly, whose developmental function has been thoroughly studied. Fruitflies have been used for over a century as a very useful experimental model to study how animals construct themselves. Most of these studies have provided important ideas and information to understand how these processes of animal construction may work in all other animals, including humans.

A major aim of modern developmental biology is to understand the mechanistic basis underlying the formation of animal structures. To achieve this, we must understand how complex programs encoded in the genome are established during development and how the activity of these programs relates to morphology and function. The Drosophila Hox gene Ultrabithorax (Ubx) controls the development of the posterior thorax and anterior abdomen. Ubx produces a family of protein isoforms through alternative splicing (AS); the isoforms differ from one another by the presence of small (micro) exons, and recent work in my lab has shown that Ubx isoforms possess distinct biological functions in vivo and in vitro. Notably, the original work describing the Ubx isoforms also reported a striking correlation between Ubx AS and polyadenylation (pA) patterns so that some of the splicing isoforms have a short 3'UTR tail while others have a longer 3'UTR. Furthermore, a recent report indicates that Ubx expression is regulated by microRNAs (miRNAs) acting via target sites located in Ubx 3'UTR. Remarkably these targets are located in sequences affected by differential pA. These observations suggest the hypothesis that the coupling between Ubx alternative splicing and differential polyadenylation is functionally related to Ubx miRNA-regulation during Drosophila development. This project will investigate the molecular underpinnings and developmental consequences of the coupling among alternative splicing, differential polyadenylation and miRNA regulation in the Drosophila Hox gene Ultrabithorax (Ubx) in vivo, studying the effects of these interactions on Ubx transcript structure, expression levels, and biological functions during Drosophila development. My work thus uses an interesting developmental example from Drosophila to inform us about the biological relevance of the general phenomenon of molecular cross-talk among the various hierarchical levels affecting gene expression control.