The Role of Long Non-Coding RNA, CCDC26, in Gene Regulation

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

The recent discovery of chromatin-associated non-coding RNAs (ncRNAs) has significantly reformed our interpretation of biological gene expression. Only 2% of the human genome encodes protein, but its gene regulatory systems are exceptionally complex. In recent years, the emergence of high-throughput gene technologies such as NGS (Next-Generation Sequencing) has enabled us to uncover novel, non-coding transcripts, many of which are not transcribed from protein-coding regions. Increasing evidence suggests that these regions, formerly referred to, as "junk DNA", constitute a new, critical layer of genomic regulation we are yet to fully understand. There are very few examples of established long non-coding RNA (lncRNA) molecular mechanisms. A large number of lncRNAs have been identified in recent years, but the vast majority have undetermined functions. One such lncRNA is RP11-419-K12. It does not appear to encode a functional protein, but possesses highly conserved regions within its introns suggesting biological function of the RNA itself. Past studies indicate a role in myeloid differentiation. Thus, we intend to begin this project by characterising RP11-419-K12 in an AML cell line, aiming to answer the key questions of its location within the cell, and what the length and sequence is of the dominant form of this transcript in this particular cell line.
The over-arching aim is to determine the function of RP11-419-K12 by applying techniques to determine its interactions, 3D contacts, and the effects of its depletion. Comparisons of results from different cell lines, along with identification of genes that may be regulated by the RNA will help establish a better understanding of transcription and lncRNA function. This could have applications in healthcare and lead to the utilisation of lncRNAs as biomarkers or drug targets.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1644225 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Rhian Jones
 
Description So far, the grant funding this research project has led to the discovery of two potential, novel functions of a particular long non-coding RNA (lncRNA) in a leukaemia cell line. Funding has allowed me to employ fluorescence in situ hybridisation (FISH) and microscopy techniques, resulting firstly in the finding that this particular RNA functions in regulating 3-dimensional nuclear and chromatin architecture, both in cis and in trans.

Recent work by our laboratory has demonstrated that a reciprocal regulatory mechanism exists between this lncRNA and a prominent oncogene. It appears that the lncRNA regulates oncogene expression and the oncogene regulates expression of the lncRNA. My work has demonstrated that this reciprocal regulation involves changes to the 3D chromatin architecture. By employing 3D FISH and subsequently calculating the distance between these two loci in 3-dimensions using a programme written in R, I observed significant changes between WT cells and a cell line in which the lncRNA of interest has been knocked out (KO) by CRISPR methods. The distance between the loci is significantly increased in the KO cells, indicating that a contact may be required for this regulatory mechanism.
This lncRNA also appears to regulate the localisation of two loci on Chr.1 and Chr.16. Applying similar techniques to those described above, I have found that typically, these loci lie preferentially at the nuclear periphery and in close spatial proximity to the lncRNA locus. Upon lncRNA KO however, this interaction is lost and the Chr1 and Chr16 loci relocate towards the nuclear centre, whilst the lncRNA locus remains at the nuclear periphery.

I have also identified a second role of this lncRNA in regulating DNA methylation. Analysis of 5-methyl-Cytosine levels revealed that in the absence of the RNA, global methylation levels fall significantly. Further investigation indicated that this is due to a change in the localisation of the methyl-transferase enzyme, DNMT1, from the nucleus to the cytosol. Total DNMT1 protein levels however are not altered and repeat experiments performed for DNMT3A and DNMT3B suggested that this effect is specific to DNMT1. DNMT1 has been previously linked to maintaining the integrity of the nucleolus, however, nucleolar structure was not significantly altered in the KO cells.

Work is currently being undertaken to determine why the subcellular localization of DNMT1 is altered in the absence of the lncRNA. Given the extent to which DNMT1 is post-translationally modified, work is currently being undertaken to assess whether any changes in phosphorylation or acetylation etc. contribute. This will shed light on whether this RNA functions in post-translational regulation of proteins.
Exploitation Route I plan to take my findings forward and determine the mechanism by which DNMT1 is being re-localised. Moreover, it will be interesting to further investigate the observed changes in methylation levels in the KO cells by large scale bisulfite sequencing. This could help us to establish cause of previously established KO cell characteristics. Finally, I would also like to assess whether there is any link between the regulation of 3-dimensional nuclear architecture and DNA methylation, or whether this lncRNA is multifunctional. I plan to begin answering some of these questions myself, and it is likely the project will be continued by another student.

The results of this investigation could contribute greatly to our knowledge of lncRNAs, an area for which it is becoming increasingly evident that we have only touched the tip of the iceberg. It could potentially provide future scientists with a general mechanism by which lncRNAs function, which could be applied to aid in the current analysis of other lncRNAs. In the more distant future, this collection of knowledge regarding lncRNAs could be applied in healthcare. The emerging importance of lncRNAs in gene expression means that these structures could provide an entirely new source of drug targets and biomarkers.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology