Accurate detection of m6A RNA modifications in native RNA sequences using third-generation sequencing | London Calling 2019
Opening this year’s London Calling conference, Eva Maria Novoa from the Center for Genomic Regulation in Spain, provided an update on her team’s work utilising nanopore sequencing to directly detect base modifications in native RNA molecules. Eva Maria started her presentation highlighting how the process of protein generation - from transcription of DNA to RNA, and subsequent translation of RNA to protein - is much more complex than we were taught at school. We now know that many more factors play a role in this process, requiring the study of the epigenome, epitranscriptome, and post-translational protein modifications. Furthermore, these factors are intrinsically linked, interacting with each other to add a further layer of complexity to functional studies.
Eva Maria showed a chart revealing that there have been significantly fewer studies of the translatome (i.e. all translated RNA molecules) than the transcriptome, which she puts down to the lack of suitable analysis technologies, rather than a lack of importance. It was originally thought that RNA modifications were a structural feature of tRNA or rRNA; however, in 2011, a publication revealed that the m6A modification, which was known to exist in mRNA was reversible. This led to the realisation that the modification may have functional properties, and, as a result, pushed the development of techniques to better analyse these modifications.
According to Eva Maria, in excess of 170 RNA different modifications have now been identified and, of these, over 70 have already been linked to human diseases, including cancer and neurological disorders. The first genome-wide method for the analysis of the modified base m6A, m6A-Seq, was published in 2012. Since this time there has been an exponential increase in the number of publications using this method and m6A has been shown to have a pivotal role in a range of cellular functions such as cell differentiation, stress response, mRNA stability, and sex determination. However, Eva Maria described how this method, which relies on traditional sequencing by synthesis (SBS) analysis, has a number of limitations for the detection of base modifications. For example, it requires the existence of selective antibodies or chemicals, which only exist for a handful of modification types and which may also exhibit cross reactivity. Further, the methodology is complex and only provides an indirect measure of modification state. It was her assertion that better methods were required. Fortuitously, a move to the Garvan Institute of Medical Research, brought her together with Martin Smith who was using nanopore sequencing for direct RNA. It was apparent that this technique may be able to solve many of the challenges faced by current technology. Eva Maria described how she was ‘super excited’ when Oxford Nanopore released the first direct RNA Sequencing Kit.
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28 май 2019