Function and Structure of RNA

Our group studies structural and functional motifs in RNA. We use a wide range of techniques to identify and characterize different types of RNA using sequence analysis, annotation, analysis of chemical structure probing and crosslinking data, secondary and tertiary structure prediction, molecular modelling, AI-based modelling methods, and molecular dynamics simulations. Our primary focus are viral genomic and subgenomic RNAs, as well as RNAs in other pathogenic processes including bacteria and cancers. We analyse RNA structure, RNA-RNA and RNA-Protein interactions and how these interactions affect diseases. Systems of interest span gastric cancer, mosquito-borne infections, Enteroviruses, Influenza, as well as marine pathogens. We are working to identify regulatory networks at the RNA level throughout human cell differentiation. Finally, we are studying the factors that influence manufacturability of RNAs for therapeutic use.

RNA structure in viral RNA genomes

Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our society. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying which folds the genome adopts at different stages is important to understand virus function. We employ techniques combining computational and high-throughput RNA structure-mapping approaches to aid in the understanding of structures within RNA virus genomes. Over the last year we made several advances in our understanding of flavivirus biology and the role the RNA genome of these viruses plays in their pathogenicity. We were able to show that specific RNA-RNA interactions with host transcripts can have pro- and antiviral effects and structural variants, even in UTRs, modulate the fitness of dengue virus. (1) RNA structure plays a crucial role in viral fitness and disrupting such structure creates attenuated viruses purely through synonymous mutations. This attenuation is stable in mosquitos and human hosts. (2) Finally, viral capsid proteins and their interactions with viral genomic RNA are crucial for packaging. Interestingly, binding sites of capsid proteins coincides with sites forming extensive RNA-RNA interactions in virions. We thus postulate that capsid protein is a key driver of the compaction the genomic RNA needs to undergo when getting loaded into nascent viral particles. (3)

Functional structures in SARS-CoV-2 variants

We investigated the RNA structure and RNA-RNA interactions of wildtype (WT) and a variety of variants of concern including alpha, beta, delta and omicron strains of SARS-CoV-2 in cells using Illumina and Nanopore platforms. We identify a number of functional structural elements within the SARS-CoV-2 genome. Proximity ligation sequencing allowed us to identify hundreds of RNA-RNA interactions within the virus genome and between the virus and host RNAs. We investigated the structural effect of single mutations, their impact on RNA-binding protein associations and long-range RNA-RNA interactions. Structure in the SARS-CoV-2 genome is concentrated into highly conserved structure hubs. These structure hubs show more local secondary structure, more long-range interactions within the genome, more interactions with host transcripts and more alternative interactions for the same sequence position.

SINGLE-CELL STRUCTURE PROFILING OF RNA

RNA structure is critical for multiple steps in gene regulation. However, how the structures of transcripts differ both within and between individual cells is unknown. Here we develop a SHAPE-inspired method called single-cell structure probing of RNA transcripts that enables simultaneous determination of transcript secondary structure and abundance at single-cell resolution. We applied single-cell structure probing of RNA transcripts to human embryonic stem cells and differentiating neurons. Remarkably, RNA structure is more homogeneous in human embryonic stem cells compared to neurons, with the greatest homogeneity found in coding regions. More extensive heterogeneity is found within 3′ untranslated regions and correlates with specific RNA-binding proteins. Overall, RNA structure profiles better discriminate cell type and differentiation stage than gene expression profiles alone. (6)

• Liao KC, Xie X, Sundstrom AKB, Lim XN, Tan KK, Zhang Y, Zou J, Bifani AM, Poh HX, Chen JJ, Ng WC, Lim SY, Ooi EE, Sessions OM, Tay Y, Shi PY, Huber RG, Wan Y. Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells. Genome Biology 24 (2023) 279.


• Chin WX, Kong HY, Zhu IXY, Teo ZY, Faruk R, Lee RCH, Ho SX, Aw ZQ, Ye B, Hou XJ, Tan AKY, Yogarajah T, Huber RG, Cai Y, Wan Y, Chu JJH. Flavivirus genome recoding by codon optimization confers genetically stable in vivo attenuation in both mice and mosquitos. PLoS Pathogens 19 (2023) e1011753.


• Boon PLS, Martins AS, Lim XN, Enguita FJ, Santos NC, Bond PJ, Wan Y, Martins IC, Huber RG. Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging. International Journal of Molecular Sciences 24 (2023) 8158.


• Penić RJ, Vlašić T, Huber RG, Wan Y, Šikić M. RiNALMo: General-Purpose RNA Language Models Can Generalize Well on Structure Prediction Tasks arXiv preprint arXiv:2403.00043 (2024).


• Wang JX, Zhang Y, Zhang T, Tan WT, Lambert F, Darmawan J, Huber RG, Wan Y. RNA structure profiling at single-cell resolution reveals new determinants of cell identity. Nature Methods 21 (2024) 411-422.