Long-read Nanopore sequencing to identify isoform-specific patterns of mRNA methylation

It has for decades been known that the building blocks, or 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 for decades. In stark contrast, the progress of research into RNA modifications has been very disappointing; this is especially unfortunate as these modifications are actually known to be more prevalent and chemically complex than those found in DNA. Further, many of the enzymes that catalyse the formation of RNA modifications have been linked with human disease, suggesting important biological roles.

A primary reason for slow progress in the field has been due to the fact that detailed studies identifying the precise positions of modifications in RNA molecules have proven 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 now beginning to realise more fully the broad scope offered by such investigations in biosciences research. The research field is however still in its early stages and a massive concerted effort is required between laboratories in order to yield necessary molecular characterizations of these RNA modifications. The studies proposed here aim to make major contributions to the field in this regard.

We have recently been developing a novel method based on long-read Nanopore sequencing for mRNA isoform-specific profiling of modification sites. Having already demonstrated proof-of-principle for such a method (see Case for Support), in this proposal we aim to show that the technique can be highly informative for isoform-specific profiling of m6A sites which cluster around stop codons of mRNAs. We will aim to confirm whether m6A methylation around stop codons of mRNAs does indeed correlate with certain patterns of alternative polyadenylation of transcripts, which was previously suggested using short-read sequencing based methods. Further, we will use cells deficient in the m6A demethylase FTO to also assess whether certain sets of isoforms are specifically targeted for dynamic regulation.

We hope that this study will serve as a platform for the development of additional similar methodologies which will aim to further characterize the 'epitranscriptome' using long-read sequencing in future works.

The Oxford Nanopore Technology platform is exciting in numerous regards with several potential practical applications being suggested. These range from uses in agriculture, basic research, and improving human health. It is however still a fledgling technology, and studies such as this and similar are needed to help improve confidence in the platform. Our study for example will include demonstrations in advances in throughput as well as accuracy of the method, and will therefore be of general interest to those who might consider using the technology.