Novel functions of alternative pre-mRNA splicing coupled with nonsense-mediated decay

Precise regulation of cell- and tissue-specific genes is indispensable for proper development and function of our organism. Importantly, abnormal gene expression has been linked to devastating neurological and neuropsychiatric diseases such as myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, Alzheimer's disease and dementias. Yet another example of potentially dire consequences caused by defective genes is provided by a range of hereditary disorders affecting multiple tissues and organs. Many of these conditions are associated with considerable patient suffering and mortality and constitute a serious burden for the national health system.

Expression of our genes proceeds as a succession of distinct molecular steps that include transcription, processing of primary transcripts into messengers, and ultimately translation of the messengers into proteins. Several control mechanisms operating at each of these steps ensure that protein products of our genes appear in the right place and at the right time. One such control called nonsense-mediated decay, or NMD, eliminates messengers with defective protein-coding capacity thus allowing the cell to cope with deleterious mutations and occasional errors in primary transcript processing. Surprisingly, recent studies suggest that this mechanism is also used to regulate gene expression under normal circumstances. In this case, corresponding primary transcripts can be processed to generate two or more alternative messenger variants, with some variants encoding proteins and the others degraded by NMD. Several genes indispensible for our nerve cells appear to be regulated in this manner and additional research in this field promises further exciting discoveries.

Here we propose to uncover novel genes that are controlled by alternative processing/NMD during brain development and to begin understanding their functional significance. We will advance our research by combining molecular, cellular and developmental biology techniques with advanced bioinformatics approaches. Our work will have important biomedical implications. Indeed, at least one novel gene, Hps1, identified in our preliminary experiments has been directly linked to a hereditary disease called Hermansky-Pudlak Syndrome and understanding regulation of this gene will likely provide important insights into this and a large group of other metabolic disorders associated with similar molecular defects. Wider medical relevance of our study will be guaranteed by its general relevance to neurological and neuropsychiatric diseases associated with aberrant regulation of genes.

An important transcriptome-wide event in differentiating neurons involves coordinated down-regulation of multiple genes expressed at high levels in neural precursors (NPs). Here we will investigate a novel mechanism mediating this transition by switching alternative splicing (AS) patterns of NP-specific transcripts and triggering their nonsense-mediated decay (NMD). We hypothesise that RNA-binding protein Ptbp1 that is expressed at high levels in NPs but down-regulated in neurons, may coordinate a large part of this AS-NMD program. Indeed, our preliminary studies have begun uncovering genes expressed at optimal level in the presence of Ptbp1 but undergoing NMD in its absence.

To further test our hypothesis, we will first identify corresponding target genes in mouse neuroblastoma cells by transcriptome-wide RNA sequencing followed by advanced bioinformatics interpretation of condition-dependent changes in the numbers of exonic, intronic and splice junction RNA-seq reads. We will validate neuroblastoma data and refine our understanding of the Ptbp1/AS-NMD network using an in vitro neurogenesis model, primary cultures of neural stem cells and neurons and ex vivo neural tube explants. Finally, we will elucidate biological significance of the most promising Ptbp1/AS-NMD targets in cell lines and primary cultures through a combination of molecular, cellular and developmental biology approaches.

By focusing on a novel class of Ptbp1/AS-NMD targets and performing differentiation stage-resolved analyses the proposed program will be poised to generate valuable insights into fundamental mechanisms underlying brain development and function. Importantly, at least one Ptbp1-activated AS-NMD target, Hps1, has been linked to a hereditary condition called Hermansky-Pudlak Syndrome. Wider medical implications of our study will be guaranteed by its general relevance to neurological and neuropsychiatric diseases associated with aberrant regulation of genes.

We anticipate that the proposed programme will generate considerable economic and societal impacts by contributing to medicine, education and staff training.

Medicine
Underscoring medical significance of our work, many human diseases are associated with defects in posttranscriptional control of gene expression. Importantly, RNA-based processes are deregulated in several devastating neurological and neuropsychiatric disorders including myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, and possibly Alzheimer's and Huntington's diseases. These conditions accompanied by considerable patient morbidity and mortality also account for an extremely large fraction of the annual £112 billion cost of brain disorders in the UK (J. Psychopharmacol. 2013, 27:761-770). By shedding light on important aspects of RNA metabolism in the nervous system context the proposed programme should improve our understanding of molecular aetiology of these and other diseases and ultimately lead to developing advanced therapies and diagnostic tools. Our work will also have another important biomedical dimension. At least one gene identified in our preliminary experiments, Hps1, has been directly linked to Hermansky-Pudlak Syndrome, a hereditary disease related to a larger cohort of lysosomal storage diseases occurring with collective incidence of ~1 in 7000-8000 live births (Biochem. Soc Trans. 2000, 28:150-154). New insights into Hps1 regulation generated by our program may eventually improve the way these diseases are detected and treated in clinical settings. Although materialisation of these important benefits may require years and possibly decades, we are convinced that contribution of our work to this process will be substantial.

Education
Our work will rely on a multidisciplinary strategy combining bioinformatics and systems biology with more traditional approaches. This trend is becoming increasingly prevalent in life sciences thus necessitating corresponding updates to secondary and tertiary science education. By reaching out to school and undergraduate students we hope to generate valuable experience that may impact future long-term changes in the education sector. Of note, a number of undergraduate students successfully completed short, typically 2-12 month research projects in PI's lab in the past. In 2015-2017, the lab is expected to host students from the Judd School (Tonbridge, Kent) and KCL undergraduates. We plan to engage these students in experimental and computational biology projects outlined in this proposal, which will provide them with first-hand experience in multidisciplinary research. We will additionally use our methodology and data as a teaching device in lectures and tutorials for KCL undergraduate and graduate students.

Staff training
The proposed project will provide a natural framework for training of a postdoctoral fellow who will master a wide range of molecular biology, cell engineering, developmental neurobiology and bioinformatics techniques and will additionally acquire advanced communication and managerial skills. This comprehensive training will maximise his or her value as a skilled employee capable of making important contributions to the UK academia and industry within 3-6 years since the start of the programme. Moreover, by interacting with other lab members the postdoctoral fellow will undoubtedly facilitate their professional growth.