In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.

Mcm1 is an essential protein in yeast and a founding member of the MADS box family of transcriptional regulatory factors, a highly conserved group of proteins found in virtually all eukaryotic organisms (38,40). Although members of this family share conserved DNA-binding and dimerization domains, they regulate a wide range of cellular functions, from basic metabolism to control of the cell cycle and determination of cell type. MADS box proteins bind with high affinity and specificity to their target genes in vitro; however, they often require accessory or cofactor proteins to specifically bind to their target sites in vivo. For example, in plants, MADS box proteins form heterodimers to regulate specific sets of genes required for flower development, and in mammals, the SRF protein interacts with several different cofactors to activate different sets of genes in response to serum stimulation (35,37,46). In many cases, the cofactor interactions of MADS box proteins determine which genes are regulated and if these genes are transcriptionally activated or repressed. Therefore, determination of how MADS box proteins interact with different cofactors to achieve target specificity and proper transcriptional regulation is essential for understanding the underlying mechanisms of regulation of many cellular and developmental processes.A simple example of a combinatorial network involving a MADS box protein is the transcriptional regulatory system that specifies cell mating type in the yeast Saccharomyces cerevisiae (16). Yeasts have two haploid cell types, a and ␣, that differ by their cell surface receptors and the pheromones they secrete. Exposure of an a or ␣ cell to the pheromone of the opposite mating type triggers a signal transduction cascade, inducing genes that are required for mating and the formation of the diploid a/␣ cell type. In a cells, the MADS box protein Mcm1 binds to target sites upstream of the promoters of a-specific genes to activate their transcription (2) (Fig. 1). In ␣ cells, Mcm1 combines with the ␣1 protein to activate transcription of ␣-specific genes (4,20,33,43). Mcm1 also combines with the ␣2 protein in ␣ cells to bind to the pheromone response element (PRE) to repress transcription of a-specific genes (23,32). In addition to determining the expression of the cell typespecific genes, Mcm1 interacts with the Ste12 protein in haploid a and ␣ cells to activate genes required for mating and cell fusion (10,12,13,19,31). The interaction of Mcm1 with its different cofactors, therefore, determines the target specificity of Mcm1 and, in the case of ␣2, also changes it from functioning as a transcriptional activator to functioning as a repressor.The crystal structure of the ternary complex of Mcm1 bound with ␣2 to DNA provides an excellent model for understanding how Mcm1 binds DNA and interacts with this cofactor (44). Comparison of the Mcm1 structure with the mammalian SRF and MEF2 proteins in complex with DNA revealed that the protein conformation and many of the DNA contacts are remarkably conse...

The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23⌬ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23⌬. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ϳ45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function.

No abstract