Rrp5 Binding at Multiple Sites Coordinates Pre-rRNA Processing and Assembly

Simon Lebaron,Åsa Segerstolpe,Sarah L French,Tatiana Dudnakova,Flavia De Lima Alves,Sander Granneman,Juri Rappsilber,Ann L Beyer,Lars Wieslander,David Tollervey

doi:10.1016/j.molcel.2013.10.017

Abstract

SummaryIn vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0–A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0–A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced.

Highlights

The yeast preribosomal RNA undergoes a complex processing and assembly pathway to generate the mature ribosomal subunits
Rrp5 Has Multiple Binding Sites on the Pre-rRNA A His6-TEV-Protein A (HTP) cassette was inserted at the C-terminal end of the RRP5 gene in strain BY4741 (Figure 1A)
The cells were harvested by centrifugation, lysed, and Rrp5-HTP was partially purified prior to crosslinking in a Stratalinker (Granneman et al, 2009)

Summary

Introduction

The yeast preribosomal RNA (pre-rRNA) undergoes a complex processing and assembly pathway to generate the mature ribosomal subunits. Three early pre-rRNA cleavages, at sites A0, A1, and A2, generate the 20S pre-rRNA, which is the direct precursor to the 18S rRNA (Figure S1 available online). These cleavages can either occur posttranscriptionally following release of the 35S pre-rRNA (released transcript cleavage; RTC) or cotranscriptionally on the nascent transcript (nascent transcript cleavage; NTC) (Kos and Tollervey, 2010; Osheim et al, 2004) The first committed step on the major pathway of 5.8S and 25S rRNA maturation is cleavage at site A3 by the RNA-protein complex ribonuclease (RNase) MRP (Figure S1). Cleavage at A3 predominately occurs posttranscriptionally, and the timing is linked to events at the 30 end of the 35S prerRNA (Allmang and Tollervey, 1998), but the nature of the linkage is unclear

Results

Discussion

Conclusion