Function and Structure of RNA

BII Function and Structure of RNA


Our group focuses on identifying structural and functional motifs in RNA. We use a wide range of techniques to identify and characterize different types of RNA, including sequence analysis, covariation, gene annotation, analysis of chemical structure probing and crosslinking data, secondary and tertiary structure prediction methods, molecular modeling, and molecular dynamics simulations. We are particularly interested in viral genomic and subgenomic RNAs including RNA-RNA and RNA-Protein interactions within infected host cells. Additionally, we are working on multi-omics datasets to identify causal factors for diseases and develop new therapies.

BII - Function and Structure of RNA Figure 1

Figure 1: Dengue Virion with RNA and Capsid Proteins. Representation of Dengue genome (orange) in complex with capsid proteins (purple) packaged into a mature virion (grey, blue). Mature virions contain an 11 kb RNA genome that exhibits complex local and long-range structure that is essential for viral function. We identified key RNA structural features even in the coding regions of the virus and showed conclusively that the RNA structure is necessary by synonymous mutagenesis which disrupted viral fitness and compensatory mutations that were able to restore viral function.

Functional Structural Elements in SARS-CoV-2 Genomes

In light of the ongoing COVID-19 pandemic the SARS-CoV-2 coronavirus has been a high priority for our group. In 2021 we published a landmark study identifying key structural elements within the viral genome and characterized the topology of the virus. Our prediction for functional sites has since been confirmed experimentally and may prove useful for the design of attenuated strains for vaccination and research. Additionally, our study for the first time investigated structural differences in the subgenomic transcripts of SARS-CoV-2. The virus produces a number of transcripts in varying abundance encoding different structural components of the virus. Using Oxford Nanopore direct-RNA sequencing, we were able to differentiate these transcripts and study their structures individually. We show that certain segments adopt unique folds that could not have been detected previously.

Development of A Sars-cov-2 Therapeutic

An infection route via binding to human ACE2 represents a common virus entry mechanism shared among at least three known human coronavirus species – HCoV-NL63, SARS-CoV-1, and SARS-CoV-2. Serving as an entry receptor, ACE2 is inherently resilient to viral escape, which makes its recombinant soluble form a potential universal therapeutic approach against all the ACE2-utilizing coronaviruses. Using a combination of computational simulation and combinatorial mutagenesis approaches, we have engineered a ACE2 variant, that showed significant improvements in both affinity and neutralization potency against the pseudo-typed SARSCoV- 1 and SARS-CoV-2. More interestingly, our engineered ACE2 potently neutralized the pseudo-typed South African (Beta, B.1.351), Indian (Kappa, B.1.617.1), and California (Epsilon, B.1.429) SARS-CoV-2 variants, which possess four mutations or more in the spike regions, along with >20 single and double mutation variants. Its neutralization activity against the UK (Alpha, B.1.1.7), Indian (Delta, B.1.617.2), and Brazilian (Gamma, P.1) variants was also retained. In November 2021 we applied for a patent for our engineered neutralizing ACE2 variant.


Figure 2: Native human ACE-2–SARS-CoV-1/2 complex. (a) The extracellular domain of ACE-2 shown in dark grey is bound to the receptor binding domain of SARS-CoV-1/2 (light grey). Image created from PDB ID 6M0J. The complex interface is outlined. (b) (top) The interface in detail. ACE-2/SARS-CoV-1 residues are coloured red and pink respectively while ACE-2/SARS-CoV-2 residues are presented in blue and light blue. Image created from PDB IDs 2AJF and 6M0J. Key residues that factor prominently in in silico SSM analysis are highlighted with bold text. (bottom left) The native interactions of ACE-2 with SARS-CoV-1 RBD consist of weak hydrophobic interactions between ACE-2 T27 and RBD L443, F460, and Y475 and several hydrogen bonds formed between the C-terminus of ACE-2’s N-terminal helix and the RBD. (bottom right) Native interactions of ACE-2 bound to SARS-CoV-2 RBD are largely similar to those of SARS-CoV-1 albeit with a greater number and distribution of hydrogen bonds and slightly more satisfied hydrophobic interactions between ACE-2 T27 and RBD F456, Y473, and Y489.

RNA Structure in Neuronal Cell Differentiation

We systematically assayed RNA structural dynamics and their relationship with gene expression, translation, and decay during human neurogenesis. We observe that the human embryonic stem cell transcriptome is globally more structurally accessible than differentiated cells and then undergoes extensive RNA structure changes, particularly in the 3’ UTRs. Additionally, RNA structure changes during differentiation are associated with translation and decay. We observed that RBP and miRNA binding is associated with RNA structural changes during early neuronal differentiation, and splicing is associated during later neuronal differentiation. Furthermore, our analysis suggests that RBPs are major factors in structure remodelling and co-regulate additional RBPs and miRNAs through structure. This study deepens our understanding of the widespread and complex role of RNA-based gene regulation during human development.

Dengue and Zika RNA-protein Interactions

In addition to intrinsic structures of the genomic RNA itself, viral RNA interacts with a variety of proteins. One of the most important interactions in the viral life cycle is the interaction of flaviviral RNA with their respective capsid proteins. These proteins are highly charged dimers with extended intrinsically disordered regions that have been shown to be crucial for their function. We investigated the structure and dynamics of these regions recently. Our current focus is on the interactions of capsid protein with genomic RNA which we are pursuing in two directions.

The first avenue of inquiry is the distribution of capsid binding along the viral genome in the packaged state. It is generally assumed that the highly charged capsid protein fulfills a histone-like function in sequestering genomic RNA. We investigated the distribution of binding along the full genome and identified hotspots that preferentially bind RNA in vitro as well as in packaged mature virions. We were able to demonstrate that the structure inside virions differs from naïve binding to in vitro transcripts. Moreover, the binding pattern is relatively specific contrary to initial expectations, with considerable preference for structured regions and avoidance of interactions involved in long-range interactions. This leads us to conclude that local structural elements likely play a role in the early association of the viral genome with membrane-associated capsid protein at the time of packaging. Long-range interactions would then be formed throughout the budding process.

Building on that hypothesis, we are interested in characterizing the binding modes that occur between RNA segments and the capsid protein. We have reason to believe that structured and unstructured RNA regions interact with distinct regions on the capsid protein. We have also used molecular dynamics simulations to demonstrate that capsid binding likely involves significant structural distortion in target RNA. Our aim regarding the identification and characterization of binding modes is mainly to explain the observed structure and sequence specificity outlined above.


Figure 3: Identifying RNA Binding Sites in Dengue. Identification of RNA-protein interactions by footprinting and crosslinking data. The genomic RNA is protected by or crosslinked with the protein segments bound at a specific location. Subsequent sequencing of the remaining products after digestion allows for the identification of these sites.


Principal Investigator HUBER Roland G.   |    [View Bio]  
Senior Post-Doctoral Research Fellow DEFALCO Louis
Senior Post-Doctoral Research Fellow DELLI PONTI Riccardo  

Selected Publications

  • Wang JX, Tong Z, Zhang Y, Wen TT, Ming W, Yang S, Lambert FRP, Huber RG, Wan Y. Genome-wide RNA structure changes during human neurogenesis modulate gene regulatory networks. Molecular Cell 81 (2021) 4942-4953.

  • Yang SL, De Falco L, Anderson DE, Zhang Y, Aw JGA, Lim XN, Tan KY, Zhang T, Chawla T, Su Y, Lezhava A, Merits A, Wang LF, Huber RG, Wan Y. Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions. Nature Communications 12 (2021) 5113.

  • Holdbrook DA, Marzinek JK, Boncel S, Boags A, Tan YS, Huber RG, Verma CS, Bond PJ. The nanotube express: delivering a stapled peptide to the cell surface. Journal of Colloid and Interface Science 604 (2021) 670-679.

  • Fleischmann J, Feichtner A, De Falco L, Kugler V, Schwaighofer S, Huber RG, Stefan E. Allosteric Kinase Inhibitors reshape MEK1 kinase activity conformations in cells and in silico. Biomolecules 11 (2021) 518.

  • De Falco L, Silva NM, Santos NC, Huber RG, Martins IC. The pseudo-circular genomes of flaviviruses: structures, mechanisms and functions of circularization. Cells 10 (2021) 642.

  • Fibrinansah G, Lim EXY, Marzinek JK, Ng TS, Tan JL, Huber RG, Lim XN, Chew VSY, Kostyuchenko VA, Shi J, Anand GS, Bond PJ, Crowe JE, Lok SM. Antibody affinity versus dengue morphology influences neutralization. PLoS Pathogens 17 (2021) e1009331.

  • View full list of publications here