Regulation of Neuronal Differentiation by Micropeptides

Understanding how tissues grow and develop is a central question in developmental biology. The nervous system is one of the most complex tissues in most animals, and contains some of the most highly specialised cell types. As with all adult tissues, the specialised cells of the nervous system are derived from a set of stem cells which can be found in developing embryos, as well as in later developmental stages, including the adult brain in humans. These neural stem cells divide in a precisely controlled manner to produce neurons, which are the functional cells of the nervous system. Much research has been done to identify genes which are activated in neurons, which in turn regulate further genes that are necessary for neuronal function. Previously, we identified a gene, lola, which acts in the opposite manner - i.e. it maintains cell's neuronal identity by switching off stem cell genes. Animals that are mutants for lola develop brain tumours which are the result of neurons reverting back to stem cells.

In an effort to understand how lola mediates these effects, we looked for further genes that interact with lola. Using this approach, we identified an interesting gene, copacobana (copa), which we believe is involved in the regulation of Lola function. In contrast to most genes, which contain the instructions required for the cell to make a large protein product, copa is divided in to a series of smaller functional units called open reading frames (smORFs), which allows this gene to make multiple small peptides. Similar small peptides have been found previously to be involved in various biological processes, however, copa is the first example of its kind to be observed in the nervous system. To understand exactly how copa is functioning in the development of neurons, we are intending to use genetic techniques to create mutant fruit flies in which copa is removed. By observing the development of the nervous system in these mutants, we will be able to determine the action of copa and its relationship to lola. The presence of smORFs within a gene is very difficult to predict. We intend to use recently developed molecular biology techniques to fully characterise the functional regions of copa, which will allow us to more fully appreciate its role in brain development.

In depth knowledge of how brain tissues are built is vital for understanding what can go wrong in congenital conditions such as microcephaly, as well as degenerative diseases such as Alzheimer's. In certain cancers, it is thought that cells undergo a reversion to a stem cell like state as observed with our lola mutants. Therefore, this project may help us to understand how that process is able to occur. We hope that this project may help to inform the development of therapeutics for these conditions and others. Furthermore, there is potential for small peptides such as the ones made by copa to be used for pharmacological or industrial purposes. smORF-containing genes are very difficult to identify, and there are currently only a handful of validated examples of this phenomenon. By identifying and characterising functional peptides which are active in the brain we are advancing our understanding of a fascinating nascent biological field. Identifying proof-of-principle of smORF action has the potential to impact almost all areas of bioscience.

Small open reading frame (smORF) encoded micropeptides have recently been shown to be a highly prevalent component of eukaryotic proteomes. Furthermore, they have been shown to have important functional roles in a variety of biological processes, however, very few examples have been well characterised. Therefore, the extent of smORF function in vivo is largely unknown. We propose to characterise a novel nervous system expressed smORF gene, which appears to have a role in the timing of neuronal differentiation.

Previously, we have shown that the transcription factor, Lola, represses the transcription of stem cell genes in mature neurons, thereby maintaining their differentiated state. Using Lola as bait in a yeast-two-hybrid protein interaction screen, we identified a novel smORF gene, which we have named copacobana (copa). We have shown that copa is highly expressed in the Drosophila larval central nervous system (CNS) and is asymmetrically segregated from neural stem cells into their progeny. Furthermore, our preliminary data suggest a mechanism in which copa encoded peptides may inhibit premature differentiation and cell cycle exit by Lola.

We propose to study the functions of copa using genetics and molecular biology approaches in Drosophila. Initially, we will study the role of the copa gene as a whole by phenotypic analysis of mutant animals. We will then investigate the function of putative copa peptides by using CRISPR/Cas9 genome editing to make precise mutations at the copa endogenous locus. As smORF-containing transcripts have frequently been shown to be poly-cistronic, (containing multiple ORFs), we will also use recently developed poly-ribosomal profiling to identify additional smORFs in the copa transcript. This research programme we will expand our knowledge of the mechanisms by which the neural development is controlled, as well as providing the first example of a smORF encoded peptide having a function in the developing nervous system.

The expected beneficiaries of the research detailed in this proposal include i) Life Scientists, ii) Businesses recruiting graduate-level staff, iii) The wider public and iv) The Life Science Industry.

Life Scientists:

The proposed research will have both specific and broad benefits to the scientific community in the UK and internationally. The work will be shared through publication, attendance and presentation at scientific meetings, and directly through the Southall lab website and other science media networks.
The biology of smORF-encoded peptides is gaining increasing amounts of attention amongst researchers across multiple disciplines. Extensive data has been presented demonstrating that smORFs and their cognate peptides are major components of eukaryotic genomes and proteomes respectively. Despite this, very few validated examples of functional smORFs in vivo have been presented, therefore the addition of copa peptides to the roster of known smORFs will significantly advance the field. Furthermore, we will be the first group to demonstrate that CRISPR/Cas9 genome editing is a useful tool for the investigation of smORFs.
The proposed work will be of great interest to researchers working in the neural development field. Much work has been done to identify the transcription factors which promote neuronal fate. However, it was not known, and had not really been considered, whether there was a mechanism to maintain differentiation after its initiation. The discovery of the role of Lola has demonstrated that there is a mechanism for maintenance of the differentiated neuronal state and also identified a key factor involved in this process. Our proposal aims to understand the mechanisms by which Lola, and therefore the maintenance of differentiation, are regulated. The general principles by which these mechanisms operate are likely to be applicable to other tissues beyond the nervous system. Therefore, this work will be of interest to the wider developmental biology community as well as neurobiologists.

Businesses recruiting graduate-level staff:

The research project will provide the opportunity for training scientists in the laboratory. This includes undergraduates (final year project and summer students), Masters students and the Research Co-Investigator. This training will ultimately prepare these individuals for highly skilled employment in the private and public sectors. The skills obtained are likely to produce individuals who will have a have a major impact on both the economy and the well-being of society.

The wider public and the Life Science Industry:

A potentially very effective way to improve physical and mental wellbeing in later life is to repair and rejuvenate tissues by instructing them to generate new cells. These cells could then restore deteriorating function to tissues by replacing or compensating for lost or damaged cells. An attractive method is to dedifferentiate post-mitotic cells, within a tissue, into a stem cell-like state and then use these stem cells to repair and rejuvenate tissues. This proposed work will provide important biological insights into how micropeptides could control the timely differentiation of cells after the introduction, or in situ generation of new stem cells in a tissue, for rejuvenating ageing tissues. In the long-term, controlled reprogramming and rejuvenation of tissues will impact the general public, improving health and well-being in the ageing population.

In recent years there has been increasing interest from the pharmaceutical and biotechnology industries for the use of bioactive small peptides in practical applications such as for therapeutic agents or pesticides. Due to the small size of such peptides they are easily synthesised and have the potential for greater tissue penetration than full length proteins. Therefore, there is a strong impetus for the discovery of peptides which have biological activity.