Characterising the human epitranscriptome using catalysis-dependent RIP-Seq approaches

It has for decades been known that the building blocks, known as bases, within DNA and RNA molecules can be chemically modified. In DNA, the occurrence and biological function of such modifications has been a fundamentally important area of biosciences research with far-reaching impact. In stark contrast, the progress for research into RNA modifications has been very disappointing which is especially unfortunate as these modifications are actually known to be more prevalent and chemically complex than in DNA; and many of the enzymes that catalyse the RNA modifications have been linked with human disease, suggesting important biological roles. The primary reason for this slow progress has been due to the fact that similar detailed studies of RNA modifications have been technically very difficult.

However in recent years, major advances in DNA/RNA sequencing techniques have been made that has allowed RNA modifications to be identified in the detail required to elucidate biological function. The first such studies were described in 2012 and indeed we are just now beginning to realise the potential scope offered by such investigations in biosciences research. The research field is however still in its very early stages and a massive concerted effort is required between laboratories in order to provide detailed maps of these RNA modifications, with preferably information regarding which cellular enzymes catalyse the modifications. The studies proposed here will provide major contributions to the field in this regard.

The epitranscriptomics field is a new exciting area of research made possible with the use of next generation sequencing methodologies (Meyer et al. 2012; Dominissini et al. 2012; Squires et al. 2012), and such studies have already enabled important biological insights to me made (Wang et al. 2014). We previously developed an experimental approach termed methylation-iCLIP (miCLIP) that could identify RNA modifications at nucleotide resolution and in an enzyme-specific fashion and which thus offers key advantages (Hussain et al, 2013b; Hussain et al. 2013c). We will use this and other techniques to yield further maps of RNA modifications catalysed by novel RNA methylases and further yield clues regarding the molecular roles using RNAseq methods. Detailed studies such as this which proposes to characterise the position-specific occurrence of RNA modifications in the transcriptome will provide the necessary (and much-needed) platform for future studies to dissect biological function in more depth.

1. Next Generation Sequencing technologies
One of the proposed benefits of 3rd generation sequencing technologies is its potential ability to directly detect nucleic acid modifications including RNA modifications. Indeed one of the front-runners in developing this novel ability is the UK-based Oxford Nanopore Technologies. The transcriptome-wide RNA modification maps generated in this study should help determine the efficiency and accuracy via which such new technologies can detect RNA modifications, and thus could potentially be very useful in helping develop and/or market this aspect of the nanopore sequencing method.

2. Human health
Many of the RNA methylase enzymes investigated in this study have been linked with human disease, however, the molecular pathogenesis is unclear largely due to a poor characterisation of RNA substrates and thus the studies proposed here would be fundamental in this regard. Examples include NSun6 which has been linked with schizophrenia and autism (Hu et al. 2011; Ripke et al. 2013); NSun5 which is deleted in the neurodevelopmental disorder Williams-Beuren Syndrome (Doll and Grzeschik 2001); and TRMT2A linked with breast cancer recurrence (Hicks et al. 2010).

Indeed my recent miCLIP-studies have revealed that the methyl-5-cytosine RNA methylase NSun3 mainly methylates RNAs encoded by the mitochondrial genome. Collaborators at the MRC Mitochondrial Biology Unit, Cambridge, have now identified NSun3 mutations in patients with a classical mitochondrial phenotype. Furthermore, they have have found that biological phenotypes can be rescued by expression of wt NSun3 in patient-derived cells.