Regulation of microRNA biogenesis from long noncoding RNAs

We have discovered unexpected properties of a recently identified class of genes, long noncoding (lnc)RNAs hosting microRNAs. In this proposal, we aim to investigate these properties in order to understand how these genes are regulated. This new knowledge will be essential for understanding normal development and diseases such as cancer, and may lead to the development of new therapeutic strategies.

The human genome is composed of very long sequences of DNA, sections of which are copied in the process of transcription to produce strands of a related molecule known as RNA. Until recently it was thought that most RNA molecules are used as templates to make proteins, which then carry out a cell's functions. However, it is now clear that we produce vast numbers of RNA molecules that do not encode proteins. These are known as noncoding RNAs and their production and function are mostly very poorly understood.

One class of noncoding RNAs that we know more about is microRNAs. Human cells produce over 2000 of these small RNAs, each of which functions to regulate expression of a particular set of target proteins by interacting with the RNA molecules that encode them. This regulation is very important in human health and disease, with many microRNAs associated with diseases such as cancer. Each microRNA is expressed in specific cell types at specific times, and it is essential for normal health and development that these expression patterns are maintained correctly.

MicroRNAs are produced by a multi-step pathway. The most important step in controlling their production is the first one, in which a long RNA molecule is recognised and cut by a molecular machine called the Microprocessor while transcription is still in progress. Almost all we know about microRNA production comes from a subset of microRNA genes that also produce protein coding RNAs. However, at least half of human microRNAs are instead located in a different type of gene, known as long noncoding (lnc)RNAs. LncRNAs are of great interest as their diverse functions in health and disease are beginning to be revealed. Exciting recent data shows that their transcription and RNA processing are different to those of protein coding genes. The consequences of this for microRNA production are currently unknown, and will be determined in this proposal.

This proposal builds on our previous work in which we identified important differences in the processing of lncRNA and protein coding microRNA genes. We identified a new mechanism of terminating the transcription process that is unique to lncRNAs hosting microRNAs. (i) We will find out how this new mechanism is controlled, showing for the first time how these two classes of gene are distinguished. We have also identified an unexpected role for an RNA processing event known as splicing in driving transcription of lncRNAs hosting microRNAs, supporting the idea that splicing is important in controlling microRNA production and that it differentially affects the two classes of microRNA genes. (ii) We will establish how splicing controls microRNA production from both lncRNA and protein coding microRNA genes. (iii) We will also determine how factors that control the process of transcription itself, and differ between these two gene classes, influence microRNA production from both.

We will use the lncRNA that hosts microRNA-122, which is biologically important in cholesterol metabolism, hepatitis C virus infection, and liver cancer, as a model to address these questions. This approach will be coupled with state-of-the art techniques to extend our analysis to all detectable microRNAs.

Together, the results of this research will give unprecedented understanding of the control of microRNA production. Understanding these control pathways gives us the potential to manipulate them, which could be very important in the future for medical treatments and biotechnology.

Control of miRNA biogenesis is crucial for normal cellular function, yet the mechanisms driving expression of individual miRNAs remain poorly understood. In this proposal, we aim to understand the biogenesis of the >50% of miRNAs that are located in long noncoding (lnc)RNAs, which our preliminary data show are processed differently to pre-mRNA miRNA host genes. The results will provide a considerable advance in our ability to understand and predict the expression of specific miRNAs, and to understand how it changes in physiological and pathological situations, while also giving new insight into the production and categorisation of lncRNAs. This research will have important implications for basic biology and for therapeutic manipulation of miRNA and lncRNA expression.

This proposal will use cutting edge molecular biology techniques to understand how miRNA biogenesis from long noncoding (lnc)RNA versus pre-mRNA host genes is controlled by transcription, splicing and chromatin environment, using the highly expressed lncRNA hosting miR-122 as a model and extending our findings by global analysis of miRNA biogenesis. We will use plasmid constructs, CRISPR modification of the endogenous gene, and an auxin-inducible degron approach to abolish miRNA processing in order to understand what drives a novel mechanism of transcription termination we have identified on lnc-pri-miRNAs. We will then establish how splicing influences pri-miRNA transcription by the NETseq approach, and miRNA biogenesis by 4-thio-uridine labelling. Finally, we will determine the effects of chromatin modfiication and RNA polymerase II C-terminal domain phosphorylation, which are known to be important in control of other cotranscriptional RNA processing events, on miRNA biogenesis.

Together, the results will provide a comprehensive understanding of the transcriptional and cotranscriptional factors that influence miRNA biogenesis from different genetic contexts.

Who will benefit?
The involvement of miRNAs in many disease processes makes manipulation of miRNA expression an important area of commercial and patient interest. Although this is a basic science proposal, the understanding of miRNA biogenesis that we achieve may open up new avenues for modulation of specific miRNAs with a role in disease processes, and will be of relevance to the biotechnology industry. In the long term it may also be important to patients with conditions in which miRNAs play a role, such as cancer and hepatitis C virus. The research will also be beneficial to the public, due to widespread interest in the human genome and its regulation, and to schoolchildren, as RNA biology is becoming increasingly important in the biosciences and medicine and is of interest to students considering studying these subjects. Finally, a postdoctoral researcher will be employed full time and an ADAC bioinformatician employed part time on this project. Their training in research and broader transferrable skills will be a significant part of the impact.


How will they benefit?
To ensure that any biotechnological and therapeutic applications of the research are fully realised, we will assess the potential for application over the course of the project and will work with the University of Nottingham's IP commercialisation office if exploitation is appropriate. The next generation sequencing datasets generated during this project will be of potential importance to researchers in industry and healthcare as well as academia and will be made publicly available.

Engagement with the public will be achieved through talks at the Café Scientifique programme in Nottingham and the local Wollaton Science club, and will also be enhanced by a new lab website. To contribute to education, we will participate in two University of Nottingham programmes: the Summer School, which is open to sixth form students from disadvantaged backgrounds, and Wonder, which is aimed at families.

The postdoctoral researcher will receive a varied training in RNA biology techniques, including next generation sequencing, and will develop specialist skills through the collaboration with the Proudfoot and West groups. The PDRA will also develop their transferable skills through numerous opportunities to present their research in local, national and international forums, through involvement in writing and reviewing manuscripts, and through outreach activities. The collaboration with ADAC will improve the collaborative skills of both the wet lab and ADAC PDRA, and will provide excellent opportunities for each to enhance their understanding of the other's field.