Abstract
Ribosome biogenesis is a conserved process in eukaryotes that requires a large number of small nucleolar RNAs and trans-acting proteins. The Saccharomyces cerevisiae MRD1 (multiple RNA-binding domain) gene encodes a novel protein that contains five consensus RNA-binding domains. Mrd1p is essential for viability. Mrd1p partially co-localizes with the nucleolar protein Nop1p. Depletion of Mrd1p leads to a selective reduction of 18 S rRNA and 40 S ribosomal subunits. Mrd1p associates with the 35 S precursor rRNA (pre-rRNA) and U3 small nucleolar RNAs and is necessary for the initial processing at the A(0)-A(2) cleavage sites in pre-rRNA. The presence of five RNA-binding domains in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Sequence comparisons suggest that Mrd1p homologues exist in all eukaryotes.
Highlights
In eukaryotes, three of the four ribosomal RNAs are derived from a single RNA polymerase I transcript, the precursor rRNA.1 Maturation of the pre-rRNA involves a series of sequential events that occur in the nucleolus [1, 2]
The MRD1 Gene Is Essential for Cell Viability—We have recently identified a gene in the dipteran Chironomus tentans (CTE314912) called Ct-RBD-12 that contains six consensus RNA-binding domains
The presence of six RBDs is unusual, and Ct-RBD-1 appears to be involved in ribosomal synthesis and/or function
Summary
Strain HKDY8, an isogenic diploid derivative of haploid strain AA255 [36], was transformed using the NotI/SalI fragment of pS002 (see Table II; plasmids are described ) containing the mrd1⌬1::hisG-URA3-hisG allele resulting in the Uraϩ strain LWDY1. PLDY141 was transformed using the NotI/SalI fragment of pS003 containing the mrd1⌬4::HIS3 allele resulting in the Hisϩ strain PLDY146. PLY647 was transformed with pS006, and Leuϩ transformants were propagated on media containing 5-FOA to obtain strain LWY008. Using genomic DNA as template, a 409-bp NotI/BamHI flanked fragment containing sequences immediately 5Ј of the initiating ATG was amplified by PCR using primers mrdD-p5A and mrdD-p5 (Table II). A PstI- and NotI-flanked fragment encoding N-terminal HA-tagged Mrd1p, amplified by PCR using primer pair Gal-MRD-5 and GalMRD-3 (Table II) and pS001 as template, was inserted into PstI-NotIrestricted B2202, creating plasmid pS006. RNA was extracted using the hot phenol method [45], and aliquots containing 40,000 cpm were electrophoresed through 1.2%
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