Our research lines include:
- Characterizing the role of RNA modifications (‘epitranscriptomics’) in human disease, synaptic plasticity and transgenerational inheritance
- Development of novel technologies and bioinformatics tools to map RNA modifications genome-wide, such as the use of direct RNA sequencing from Oxford Nanopore Technologies
- Single molecule RNA folding dynamics
- Characterization of the role, activity and regulators of specialized ribosomes in mammalian species
- Functional characterization of the unknown epitranscriptome
RNA modifications expand the RNA lexicon and are a fundamental component of RNA functionality. This code may not only enable or enhance specific chemistries in RNA-catalysed or RNA-dependent reactions, but also changes to RNA structure-function relationships that may alter signalling pathways, offering a layer of control to enable protein synthesis to be regulated in a space-, time- and signal-dependent manner.
Recently, advances in next-generation sequencing technologies have revealed the central role that these marks play in major cellular processes, such as splicing, cell fate transition or embryogenesis. Unfortunately, the limited availability of antibodies and chemicals selective to RNA modifications has so far limited our transcriptome-wide view to only a handful of RNA modifications. Consequently, the abundance, location, and function of the majority of RNA modifications remains unknown.
(Illustrations adapted from: Novoa et al., Nat Rev Mol Cell Biol 2017; Imanishi et al., Chem Communic 2017; Li et al., Nature Methods 2017; Hauenschild et al., Nucl Acids Res 2015; Stoecklin and Diederichs, EMBO J 2014)
In our lab, we are currently employing direct RNA sequencing from Oxford Nanopore Technologies (ONT), as a tool to produce high-resolution genome-wide maps of RNA modifications. In contrast to current methods used to detect RNA modifications genome-wide (RIP-Seq, Chem-Seq), ONT direct RNA sequencing is capable of detecting RNA modifications with single nucleotide resolution, in a quantitative manner, and in single molecules.
We aim to expand our understanding of the human epitranscriptome, and more specifically to decipher:
- their function and evolution
- the effects of their dysregulation in disease
- their roles brain plasticity
- their roles in transgenerational inheritance
Disentangling the role of RNA modifications in human disease
From the battery of over > 100 known RNA modifications, more than a dozen have already been linked to human diseases, including neurological disorders and cancer, highlighting their importance in proper cellular functioning. Several RNA modifications are highly enriched in brain, and mutations in several RNA methyltransferases have been associated with intellectual disability in humans and impaired cognitive ability. Unfortunately, the limited availability of antibodies and chemicals selective to RNA modifications has so far limited our transcriptome-wide view to only a handful of RNA modifications.
Using a combination of well-established cutting-edge experimental techniques, knockout mouse models and bioinformatics, we aim to analyse the distribution of certain modifications in human and mouse, to understand how dysregulation of RNA modifications lead to human diseases such as intellectual disability, cancer and infertility.
(Illustration from: Jonkhout N, Tran J, Schonrock N, Smith MA, Mattick JS and Novoa EM. The RNA modification landscape in human disease. RNA 2017)
- Development of novel technologies and bioinformatics tools to map RNA modifications genome-wide
Currently, Sequencing-By-Synthesis (SBS) technologies, such as those developed by Illumina Corp., dominate genomic research. SBS platforms operate by DNA synthesis, copying a template strand, to cyclically incorporate individual fluorescently-labelled nucleotides that are captured with an optical device. Despite the major achievements of SBS technology, its major limitation is that it is typically blind to DNA and RNA modifications. Consequently, indirect methods (RIP-Seq, Chem-Seq) are required to identify RNA modifications genome-wide.
To overcome these limitations, we propose the application of two technologies to map RNA modifications:
a) Phage Display Technologies (PDT) as a novel tool to produce antibodies against RNA modifications, where antibodies are generated by multiple rounds of antigen selection and competitor counter-selection.
b) Direct sequencing of RNA molecules using Oxford Nanopore Technologies (ONT), which employs thousands of membrane-embedded protein nanopores coupled to highly sensitive ammeters that measure ionic current passing through the pore. This technology avoids the reverse-transcription step during the library preparation, and thus can detect and measure RNA modifications from the RNA molecules that are being sequenced, in a quantitative manner, and with single nucleotide resolution.
To properly basecall RNA modifications from ONT output data, we are using a variety of tools, including machine learning, that can help us maximize the accuracy of detection of RNA modifications.
(Illustration from: Novoa EM, Mason CE, Mattick JS. Charting the unknown human epitranscriptome. Nature Rev Mol Cell Biol 2017)
- Specialized ribosomes as a novel post-transcriptional regulatory layer to quickly tune the proteome
Ribosomes have been historically thought as uniform entities, however, recent evidence suggests that its composition might be dynamically regulated, and consequently, its activity (i.e., selective translation of subsets of transcripts).
In our lab, we are using a combination of molecular biology, mouse knockouts, genome-wide high-throughput techniques (i.e. RiboSeq, FracSeq, RNASeq) and bioinformatics, to decipher which are the forces that tune mammalian ribosome composition, and how its changes across tissues, developmental stages and environmental conditions, may affect cellular function.