Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly
The CRAC UV crosslinking technique identified numerous 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 the sequence flanking A3 and to the RNA component of RNase MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural protein factors and NTPases. A key role in coordinating pre-ribosomal assembly and processing was confirmed by "Miller" chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and pre-ribosome compaction greatly reduced. Overall design: CRAC results are provided for four individual samples: Rrp5 Full-length (FL) in vivo, Rrp5 Full-length (FL) in vitro, Rrp5 NTD and Rrp5 CTD.