Oral Presentation 41st Lorne Genome Conference 2020

Moving beyond RNA sequence: uncovering the functional role of RNA structure (#29)

Vincent DA Corbin 1 2 3 , Phillip J Tomezsko 3 4 , Paromita Gupta 3 , Harish Swaminathan 3 , Margalit Glasgow 3 5 , Sitara Persad 3 5 , Matthew D Edwards 5 , Lachlan Mcintosh 1 2 , Ann Emery 6 , Ronald Swanstrom 6 , Trinity Zang 7 , Tammy CT Lan 3 , Paul Bieniasz 7 , Daniel R Kuritzkes 4 8 , Athe Tsibris 4 8 , Anthony T Papenfuss 1 2 9 , Silvi Rouskin 3
  1. Bioinformatics, Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. University of Melbourne, Melbourne, VIC, Australia
  3. Whitehead Institute of Biomedical Research, Cambridge, MA, USA
  4. Harvard Medical School, Boston, Massachussetts, USA
  5. Massachussetts Institute of Technology, Cambridge, Massachussetts, USA
  6. University of North Carolina, Chapel Hill, North Carolina, USA
  7. The Rockefeller University, New York City, New York, USA
  8. Brigham and Women's Hospital, Boston, Massachussetts, USA
  9. Peter MacCallum Cancer Center, East Melbourne, VIC, Australia

Previous work in RNA analysis has focused on RNA sequencing and expression, however RNA actually derives much of its functionality from structure. An aspect that has been poorly studied is the capacity of a given RNA sequence to assume more than one secondary structure, and how crucial this is for RNA function. 

We have used DMS-MaPseq to probe the structure of RNA in cells, developing an algorithm called ‘Detection of RNA folding Ensembles using Expectation-Maximization’ (DREEM), which can reveal dominant and alternative conformations assumed by the same RNA sequence. Previous attempts to analyse RNA structure were limited to the derivation of a population average, whereas our method is capable of revealing the widespread heterogeneous nature of RNA structure, revealing the assumption of a single average structure as incorrect.

Human immunodeficiency virus-1 (HIV-1) is a retrovirus with a 10-kb single-stranded RNA genome. HIV-1 must express all of its gene products from the same primary transcript, which undergoes alternative splicing to produce diverse protein products, including structural proteins (Gag, Pol, and Env) and regulatory factors (Tat, Rev, Vif, Vpu, Nef and Vpr). Despite the critical role of alternative splicing, the mechanisms driving splice-site choice are poorly understood, as HIV-1 does not encode any of its own splicing factors. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements. 

In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev Responsive Element (RRE) exists in cells, we discovered alternative splice site conformations which influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides conclusive evidence for the long-standing hypothesis that RNA folding regulates splice site usage, and of most significance, uncovers a major role for RNA conformation heterogeneity in regulating RNA gene expression.