Nucleic Acids Research, 2001, Vol. 29, No. 24 5001-5008
© 2001 Oxford University Press
All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae
Department of Biochemistry and Molecular Biology, IMBW, BioCentrum Amsterdam, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27SB precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27SB precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A0, A1 and A2. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.
* 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
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