Understanding Nuclear RNA Quality Control in Mammalian Nervous System

Expression of our genes proceeds through several carefully orchestrated steps including transcription and processing of primary transcripts into mature messenger molecules, export of the messengers from the nucleus to the cytoplasm and their translation into corresponding protein products. This elaborate process is required for precise regulation of cell- and tissue-specific gene expression programmes, which, in turn, is indispensable for proper development and function of the organism. Conversely, defects in gene expression processes have been linked to several human diseases including neurological and neuropsychiatric conditions such as myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, and possibly Alzheimer's disease. These devastating disorders are associated with considerable patient suffering and mortality as well as a serious burden for the national health system.

Notably, cells employ quality control (QC) mechanisms that can minimise deleterious effects of erroneous gene expression by identifying and eliminating defective messenger molecules. An important but poorly understood branch of the cellular QC prevents export of incompletely processed gene transcripts containing intervening intron sequences from the nucleus to the cytoplasm thus preventing their translation into aberrant proteins. Transcripts retained by this mechanism in the nucleus are eventually eliminated. Previous studies by our group and others have suggested that, in addition to its error surveillance role, this nuclear retention and elimination (NRE) pathway may function in the context of developing nervous system (NS) as a regulatory mechanism enabling deterministic control of important neuronal genes. However, molecular mechanisms underlying NRE in human and animal cells in general and NS cells in particular are poorly understood.

To this end, we propose to investigate mammalian NRE machinery using a combination of candidate and unbiased discovery approaches. We additionally propose to examine developmental changes in the NRE apparatus and their biological consequences in the NS context. To advance these lines of research, we will use a combination of established molecular, cellular and developmental biology techniques and will additionally develop innovative experimental approaches. We are convinced that the proposed work will shed light on molecular mechanisms ensuring accuracy of our gene expression programme and begin uncovering novel gene regulation mechanisms contributing to NS development and function. By elucidating these important aspects the proposed work should also improve our understanding of molecular causes leading to NS diseases and ultimately pave the way to new knowledge-based therapies and diagnostic tools.

Gene expression in eukaryotes involves several RNA processing and trafficking steps that contribute to structural and functional complexity in this domain of life. However, complexity comes with thermodynamically inevitable penalty of errors that must be identified and corrected. An important but poorly understood quality control mechanism operating in eukaryotic nucleus hinders export of intron-containing transcripts to the cytoplasm thus limiting their translation into aberrant proteins. Retained transcripts that fail to complete splicing within a biologically meaningful time-frame are eventually cleared by nuclear RNA degradation enzymes. Studies by our group and others suggest that, in addition to surveillance of RNA processing errors, this nuclear retention and elimination (NRE) pathway functions in the context of developing nervous system (NS) as a post-transcriptional mechanism enabling deterministic control of important neuronal genes. However, the molecular mechanisms underlying NRE in mammalian cells in general and NS cells in particular are poorly understood. Here we propose a systematic study that will examine mammalian NRE machinery using candidate and unbiased approaches and investigate developmental changes in the NRE apparatus and their functional consequences in the NS. To develop this programme, we will use a combination of established molecular, cellular and developmental biology techniques and will additionally develop innovative tools for rapid transgenesis in mammalian cells. The proposed work should shed light on molecular mechanisms ensuring quality of mammalian transcriptomes and begin uncovering novel gene expression strategies that allow developing neurons to acquire and maintain their unique cellular identity. This may ultimately improve our understanding of NS diseases linked with defective RNA metabolism and lead to new therapies and diagnostics.

We anticipate that the proposed programme will generate considerable economic and societal impact by contributing to medicine, biotechnology, 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. 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.

Biotechnology
The proposed programme will additionally deliver innovative technologies that will likely become general research tools for academia and biotech industry alike. Two specific implementations of our HILO-RMCE transgenesis platform allowing transgene co-expression applications and integration of extremely long transgenes at a predefined genomic locus should facilitate a variety of cell engineering projects in both scientific and industrial sectors. We anticipate that these impacts will be felt within 2-3 years from the beginning of programme. In a longer term (3-10 years), we will work together with KCL Business and Innovation team to establish industrial collaborators which will enable translation of newly generated intellectual property into R&D products and, as a result, more tangible benefits for the UK economy.

Education
Our work will rely on a multidisciplinary strategy combining bioinformatics, systems and synthetic 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 2014-2016, 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 framework for training a postdoctoral fellow, a research technician, and a PhD student who is expected to join PI's lab in 2014-2015. These full-time lab members will master a wide range of molecular biology, biochemistry, cell engineering, developmental neurobiology and bioinformatics techniques and will additionally acquire advanced communication and managerial skills. This comprehensive training will maximise their value as skilled employees capable of making important contributions to the UK academia and industry within 3-6 years since the start of the programme.