Despite the fact that there are over 100 different types of modifications that can occur on RNA, little is known about their function and impact on the life cycle of RNA. Pseudouridine and N6-methyladenosine are two of the most abundant RNA modifications, and although they were discovered decades ago, little is known about the factors governing which precise substrates are subjected to the modifications. The development of a sequencing method that combines dimethyl sulfate (DMS) labelling of non-structured RNAs with RNA sequencing provides transcriptome-wide information on RNA secondary structure in yeast. We hypothesized that RNA secondary structure might play a role in guiding catalysis of RNA modifications at specific sites. To test this hypothesis, we created a pipeline that allows examination of secondary RNA structures in relation to the location of modified nucleotides. The pipeline extracts secondary structure in a window of 200 nt centered around modified nucleotides, and allows studying the secondary structure either at a per-modification level or across all modified sites (‘meta-modification analysis’) while employing sequence and expression matched controls to account for sequence-level artifacts. We initially found a strong secondary structure for pseudouridine, which we subsequently determined to reflect a sequence-dependent artifact, and analysis of structure in the context of N6-methyladenosine showed a preference in methylation on non-structured RNA molecules. Our analysis sheds new light on longstanding questions regarding the factors governing RNA modification substrate specificity.
PROJECT: Transcriptome-wide exploration of the relationship between RNA secondary structure and RNA modifications
As a returning student to the Broad, I was expecting a continuation of the intensive research environment I was already familiar with, but by the end of the summer I learned that research is not enough to succeed in science. With its focus on communication, working in teams and critical constructive feedback, this program has given me the additional tools necessary to do so.