Duss Group

Assembly mechanisms of protein-RNA complexes at the single-molecule level

The Duss group uses single-molecule fluorescence microscopy in combination with integrative structural biological and biochemical approaches to understand how protein-RNA complexes are assembled and work as macromolecular machines, providing new opportunities to fight diseases and to create new functional molecular assemblies.


Previous and current research

The assembly of protein–RNA complexes (ribonucleoproteins; RNPs) is one of the most fundamental processes of all life forms, underlying transcription, translation, and splicing. Errors in RNP assembly underlie many human diseases. The formation of an RNP complex involves synthesis and correct folding of the individual protein and nucleic acid components and specific intermolecular interactions between them. A major barrier to understanding RNP assembly has been the reliance on in vitro studies based on preformed RNA and protein molecules, whereas in cells many such complexes form on the nascent RNA while it is emerging from the RNA polymerase. Furthermore, our understanding is heavily based on compositional and structural knowledge of the mature RNPs, but we are lacking structural and, importantly, dynamic information on transient biochemically unstable assembly intermediates, which are required for a full quantitative description of the assembly processes. 

We are studying the molecular mechanisms by which various RNPs assemble, and in particular are establishing how their assembly is coupled to transcription and folding of the RNA and how it is affected by other cellular factors (Fig. 1). We focus on bacterial ribosome assembly (e.g. Duss et al., Cell 2019) and on the coupling between mRNA transcription and translation (e.g. Duss et al., Nature 2014), in E. coli and the human pathogen Mycobacterium tuberculosis. Efforts are also directed towards understanding eukaryotic RNP assemblies. By using single-molecule methods we can directly watch how single protein–RNA complexes assemble in real time (Fig. 2). By combining this with high-resolution structural information on assembly intermediates, we obtain a full molecular understanding of how protein–RNA complexes are formed and how their assembly is regulated. This understanding gives us new opportunities to interfere with RNP assembly to fight a variety of diseases and to create new functional assemblies for synthetic biology.

Figure 1: The formation of protein–RNA complexes is complicated and heterogeneous, and involves several coupled processes such as transcription, nascent RNA folding, RNA processing, RNA modification, protein and small molecule ligand binding, and the action of transiently binding assembly factors.
Figure 2: Using multicolour single-molecule fluorescence microscopy, several processes occurring during protein–RNA complex assembly can simultaneously be visualised in real-time, which allows us to dissect the complicated molecular mechanisms of intrinsically heterogeneous assembly processes. Figure adapted from Duss et al., Cell 2019, and Duss et al., Nat Commun 2018.

Future projects and goals

  • Obtaining a quantitative and dynamic understanding of how the transcription machinery and cellular assembly factors (e.g. RNA modification and RNA processing enzymes) cooperate to guide ribosome assembly.
  • Investigating how bacterial non-coding RNAs, RNA binding proteins, and small molecule ligands regulate the coupling between the mRNA transcription and translation machineries, and developing new antimicrobials targeting these processes to fight infectious diseases and address our global antibiotic resistance threat.
  • Generating a dynamic molecular understanding of how RNA drives phase separation during the formation of membraneless organelles and how these dynamic states eventually turn into pathologic aggregates implicated in several neurodegenerative diseases.
  • Developing new single-molecule fluorescence microscopy methods to track nascent RNA folding and RNP assembly.