Poster abstracts

Poster number 79 submitted by Xin Liu

RNA Chaperone Activity in Ribosome Biogenesis

Xin Liu (Department of Biochemisty and Molecular Biology, Rosalind Franklin University), Binal N. Shah (Department of Biochemisty and Molecular Biology, Rosalind Franklin University), Carl C. Correll (Department of Biochemisty and Molecular Biology, Rosalind Franklin University)

Abstract:

During ribosome biogenesis, structural rearrangements in the pre-rRNA and in trans acting snoRNAs are thought to help the small subunit (SSU) processome coordinate the sequential cleavage events that liberate the SSU precursor. Cleavage or protein binding may trigger structural changes that transition the RNA from one state to the next, thereby permitting the next step in processing or assembly. Here we focus on how a U3 specific protein Imp3p mediates one of the three sites of hybridization between the pre-rRNA and U3 snoRNA, which is required for the pre-rRNA cleavages (A1 and A2) that release the 20S small subunit rRNA precursor. In contrast to two of the U3-pre-rRNA duplexes, the third, designated the U3-18S duplex, is only observed in the presence of protein using minimal substrates in vitro. Duplex formation is blocked because prior to hybridization bases from both duplex strands are buried: one in an A/A’ stem-loop structure in U3 snoRNA and the other in helix 1 (H1) of the 18S region of the pre-rRNA. To test whether protein binding removes hybridization barriers by unfolding relevant stem-loop structures in the U3 and pre-rRNA we assessed base accessibility by chemical probing with CMCT in the presence and absence of protein. We show that Imp3p alone is sufficient to unfold the stable U3 structure and to mediate rapid U3-18S hybridization. Moreover, we show that Imp3p binds to a 257 nt pre-rRNA fragment containing H1. Addition of Imp3p results in a marked increase in accessibility of the bases in H1 involved in U3-18S hybridization and bases that flank the A1 cleavage site. The increased base accessibility of H1 provides evidence that Imp3p binding unfolds this stem structure, presumably to expose bases for U3-18S duplex formation, just as Imp3p binding unfolds the U3 stem structure. Perhaps the late arrival of Imp3p during assembly of the SSU processome mediates U3-pre-rRNA hybridization to signal subsequent A1 (and possibly A2) cleavage.

Keywords: Chaperone, Conformational Change, Ribosome biogenesis