Demonstration of protein cooperativity mediated by RNA structure using the human protein PUM2 (original) (raw)

  1. Inga Jarmoskaite2,
  2. Pavanapuresan P. Vaidyanathan2,
  3. William J. Greenleaf3,4,5 and
  4. Daniel Herschlag2,6
  5. 1Program in Biophysics, Stanford University, Stanford, California 94035, USA
  6. 2Department of Biochemistry, Stanford University, Stanford, California 94035, USA
  7. 3Department of Genetics, Stanford University, Stanford, California 94035, USA
  8. 4Department of Applied Physics, Stanford University, Stanford, California 94035, USA
  9. 5Chan Zuckerberg Biohub, San Francisco, California 94158, USA
  10. 6Departments of Chemical Engineering and Chemistry, Stanford University, Stanford, California 94305, USA
  11. Corresponding author: herschla{at}stanford.edu

Abstract

Posttranslational gene regulation requires a complex network of RNA–protein interactions. Cooperativity, which tunes response sensitivities, originates from protein–protein interactions in many systems. For RNA-binding proteins, cooperativity can also be mediated through RNA structure. RNA structural cooperativity (RSC) arises when binding of one protein induces a redistribution of RNA conformational states that enhance access (positive cooperativity) or block access (negative cooperativity) to additional binding sites. As RSC does not require direct protein–protein interactions, it allows cooperativity to be tuned for individual RNAs, via alterations in sequence that alter structural stability. Given the potential importance of this mechanism of control and our desire to quantitatively dissect features that underlie physiological regulation, we developed a statistical mechanical framework for RSC and tested this model by performing equilibrium binding measurements of the human PUF family protein PUM2. Using 68 RNAs that contain two to five PUM2-binding sites and RNA structures of varying stabilities, we observed a range of structure-dependent cooperative behaviors. To test our ability to account for this cooperativity with known physical constants, we used PUM2 affinity and nearest-neighbor RNA secondary structure predictions. Our model gave qualitative agreement for our disparate set of 68 RNAs across two temperatures, but quantitative deviations arise from overestimation of RNA structural stability. Our results demonstrate cooperativity mediated by RNA structure and underscore the power of quantitative stepwise experimental evaluation of mechanisms and computational tools.