Nucleic Acids Research, 2002, Vol. 30, No. 19 4222-4231
© 2002 Oxford University Press
Deletions in the S1 domain of Rrp5p cause processing at a novel site in ITS1 of yeast pre-rRNA that depends on Rex4p
Faculty of Science/Division of Chemistry, Department of Biochemistry and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
*To whom correspondence should be addressed. Tel: +31 20 444 7545; Fax: +31 20 444 7553; Email: raue{at}chem.vu.nl
Present address:
Jaap Venema, Solvay Pharmaceuticals, Department of Biotechnology, C.J. van Houtenlaan 36, 1382 CP Weesp, The Netherlands
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Rrp5p is the only protein so far known to be required for the processing of yeast pre-rRNA at both the early sites A0, A1 and A2 leading to 18S rRNA and at site A3, the first step specific for the pathway leading to 5.8S/25S rRNA. Previous in vivo mutational analysis of Rrp5p demonstrated that the first 8 of its 12 S1 RNA-binding motifs are involved in the formation of the ‘short’ form of 5.8S rRNA (5.8SS), which is the predominant species under normal conditions. We have constructed two strains in which the genomic RRP5 gene has been replaced by an rrp5 deletion mutant lacking either S1 motifs 3–5 (rrp5-
3) or 5–8 (rrp5-4). The first mutant synthesizes almost exclusively 5.8SL rRNA, whereas the second one still produces a considerable amount of the 5.8SS species. Nevertheless, both mutations were found to block cleavage at site A3 completely. Instead, a novel processing event occurs at a site in a conserved stem–loop structure located between sites A2 and A3, which we have named A4. A synthetic lethality screen using the rrp5-3 and rrp-4 mutations identified the REX4 gene, which encodes a non-essential protein belonging to a class of related yeast proteins that includes several known 3'
5' exonucleases. Inactivation of the REX4 gene in rrp5-3 or rrp-4 cells abolished cleavage at A4, restored cleavage at A3 and returned the 5.8SS:5.8SL ratio to the wild-type value. The sl phenotype of the rrp5/rex4– double mutants appears to be due to a severe disturbance in ribosomal subunit assembly, rather than pre-rRNA processing. The data provide direct evidence for a crucial role of the multiple S1 motifs of Rrp5p in ensuring the correct assembly and action of the processing complex responsible for cleavage at site A3. Furthermore, they clearly implicate Rex4p in both pre-rRNA processing and ribosome assembly, even though this protein is not essential for yeast.
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