Structural Probing with MNase Tethered to Ribosome Assembly Factors Resolves Flexible RNA Regions within the Nascent Pre-Ribosomal RNA

Abstract

The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13.