Structural biology of the mRNA interactome and TRIM proteins

• Applicant: Professor Dr. Janosch Hennig
• Subject Area: Structural Biology ; Biophysics ; Developmental Biology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 267437786

• Final Report Year: 2022

Final Report Abstract

The two major objectives of this proposal were to investigate the connection between ubiquitination and RNA binding in TRIM proteins and how novel RNA binding proteins/domains recognize their cognate RNA motifs. Both aims have a focus on a mechanistic understanding, obtained by combining a range of structural biology methods, including NMR, X-ray crystallography, small-angle X-ray and neutron scattering. For the second objective, we could reach the goal as described above, which is an essential step to fully understand the involvement of this protein in neuronal development. In this instance we could again demonstrate how powerful integrative structure modelling is. We also demonstrated how useful NMR can be in the validation of potential novel RNA binding proteins identified in mRNA interactome capture studies. Interestingly, we could confirm RNA binding for some but not all newly identified RNA binding proteins. Regarding the first objective, we are on our way towards establishing a general understanding of the TRIM2/3-related connection between RNA binding and ubiquitination. We are not yet at a stage where we can summarize our findings into a coherent article with high impact, but due to recent results, we are very much optimistic that this can be done within the next year. A surprising turn of events occurred during our work on TRIM25. Also here, we expected that RNA binding will initiate ubiquitination of the adjacent mRNP complex to regulate mRNA translation or degradation. However, based on our structural, biophysical and biochemical results we had to revise our hypothesis and now try to proof that RNA binding will actually increase efficiency of TIRM25 mediated ubiquitination.

Publications

• (2015). The crystal structure of the NHL domain in complex with RNA reveals the molecular basis of Drosophila braintumor-mediated gene regulation. Cell Reports 13(6): 1206-1220
  Loedige I, Jakob L, Treiber T, Ray D, Stotz M, Treiber N, Hennig J, Cook KB, Morris Q, Hughes TR, Engelmann JC, Krahn MP, Meister G
  
• (2017). Segmental, domain-selective perdeuteration and small-angle neutron scattering for structural analysis of multi-domain proteins. Angewandte Chemie Int Ed Engl 56(32): 9322-9325
  Sonntag M, Jagtap PKA, Simon B, Appavou MS, Geerlof A, Stehle R, Gabel F, Hennig J, Sattler M
  
• (2018). A general small-angle X-ray scattering-based screening protocol validated for protein-RNA interactions. ACS Combinatorial Science 20(4): 197-202
  Chen PC, Masiewicz P, Rybin V, Svergun D, Hennig J
  
• (2018). Molecuar mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition. Nature Communcations 9(1): 1820
  Koliopoulos MG, Lethier M, van der Veen AG, Haubrich K, Hennig J, Kowalinski E, Stevens RV, Martin SR, Reis E Sousa C, Cusack S, Rittinger K
  
• (2018). The role of small-angle scattering in structure-based screening applications. Biophys Rev 10(5): 1295-1310
  Chen PC, Hennig J
  
• (2019). Combined Small-Angle X-ray and neutron scattering restrains in molecular dynamics simulations. J Chem Theory Comput 15(8):4687-4698
  Chen PC, Shevchuk R, Strnad FM, Lorenz C, Karge L, Gilles R, Stadler AM, Hennig J, Hub JS
  
• (2019). Emerging RNA-binding roles in the TRIM family of ubiquitin ligases. Biol Chem 400(11):1443-1464
  Williams FP, Haubrich K, Perez-Borrajero C, Hennig J
  
• (2020). Integrative Structural Biology of protein-RNA complexes. Structure 28(1): 6-28
  Dimitrova-Paternoga L, Jagtap PKA, Chen PC, Hennig J
  
• (2020). Structure-based screening of binding affinities via small-angle X-ray scattering. IUCrJ 7: 644-655
  Chen PC, Masiewicz P, Perez K, Hennig J
  
• (2021). Mechanistic insights into RNA binding and RNA-regulated RIG-I ubiquitination by TRIM25
  Haubrich K, Augsten S, Alvarez L, Huppertz I, Simon B, Perez K, Masiewicz P, Lethier M, Rittinger K, Gabel F, Hentze MW, Cusack S, Hennig J
  
• (2021). Structure and dynamics of the quaternary hunchback mRNA translation repression complex. Nucleic Acids Res 49(15): 8866-8885
  Macošek J, Simon B, Linse JB, Jagtap PKA, Winter SL, Foot J, Lapouge K, Perez K, Rettel M, Ivanović MT, Masiewicz P, Murciano B, Savitski MM, Loedige I, Hub JS, Gabel F, Hennig J
  
• (2021). Validation and classification of RNA binding protein identified by mRNA interactome capture. RNA 27(10): 1173-1185
  Vaishali, Dimitrova-Paternoga L, Haubrich K, Sun M, Ephrussi A, Hennig J