Two‐hybrid Mpp10p interaction‐defective Imp4 proteins are not interaction defective in vivo but do confer specific pre‐rRNA processing defects in Saccharomyces cerevisiae

Published online February 27, 2004 1404±1413 Nucleic Acids Research, 2004, Vol. 32, No. 4 DOI: 10.1093/nar/gkh318 Two-hybrid Mpp10p interaction-defective Imp4 proteins are not interaction defective in vivo but do confer speci®c pre-rRNA processing defects in Saccharomyces cerevisiae Jennifer E. G. Gallagher1 and Susan J. Baserga1,2,3,* 1 Department of Genetics, 2Department of Molecular Biophysics and Biochemistry and 3Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520-8024, USA Received January 26, 2004; Revised and Accepted February 6, 2004 The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of the U3 snoRNA and at least 28 protein components, that is required for biogenesis of the 18S rRNA. We tested the function of one protein±protein interaction in the SSU processome, Mpp10p±Imp4p, in ribosome biogenesis. Exploiting the reverse twohybrid system, we screened for mutated Imp4 proteins that were conditionally defective for interaction with Mpp10p. Three different imp4 sequences were isolated that: (i) conferred conditional growth in the two-hybrid strain; (ii) complemented the disrupted imp4; (iii) conferred conditional growth in the context of their normal cellular function; and (iv) resulted in defective pre-rRNA processing at the non-permissive temperatures. Domain swapping revealed that mutations that conferred cold sensitivity resided in the N-terminal coiled-coil domain while mutations in the C-terminus conferred temperature sensitivity. Surprisingly, the mutated Imp4 proteins were not measurably defective for interaction with Mpp10p in the context of the SSU processome. This suggests that other members of the complex may contribute to maintaining the Mpp10p±Imp4p interaction in this large RNP. Since protein±protein interactions are critical for many different aspects of cellular metabolism, our work has implications for the study of other large protein complexes. INTRODUCTION In eukaryotes, ribosome biogenesis requires the coordination of many different events, including rRNA transcription, prerRNA modi®cation and processing, ribosomal protein production and rRNA±ribosomal protein assembly. Pre-rRNA modi®cation occurs early in ribosome biogenesis on the RNA polymerase I-transcribed nascent pre-rRNA (1). In Saccharomyces cerevisiae this 35S rRNA bears three rRNAs (18S, 5.8S and 25S), which are subsequently released from their nascent transcripts by pre-rRNA cleavages (Fig. 1). The U3 snoRNA and its associated proteins form a large ribonucleoprotein (RNP), the SSU processome, which is required for the three cleavage events that mature the 18S rRNA (A0, A1 and A2) (3). The SSU processome is associated with the 35S and 23S pre-rRNAs, both of which retain the U3-dependent cleavage sites (4). U3 snoRNA±pre-rRNA base pairing is required for cleavage, and this likely occurs in the context of the SSU processome (5,6). Mpp10p, Imp4p and Imp3p are three protein components of the SSU processome that are essential for its function. Mpp10p was originally discovered in a screen for human proteins that are phosphorylated during mitosis (7). Mpp10p in both humans and yeast is speci®cally associated with the U3 snoRNA, and is required for pre-rRNA processing (8,9). Mpp10p bears multiple protein±protein interaction domains (coiled-coil) and indeed has been found to interact with two proteins in a two-hybrid screen and in vivo, Imp3p and Imp4p (10). We have hypothesized that Imp3p contacts the U3 snoRNA directly via its ribosomal protein S4 RNA binding motif. Imp4 is the founding member of the Imp4 superfamily, and as such bears the s70-like RNA binding motif (4). Like Mpp10p, both Imp3p and Imp4p are U3 snoRNA-associated and are required for pre-rRNA processing (10). In a large RNP with 28+ proteins like the SSU processome, it is likely that multiple protein±protein interactions occur and that they are essential for SSU processome function. We tested the role of one of these protein±protein interactions, Mpp10p± Imp4p, in ribosome biogenesis. We used the reverse two-hybrid system approach to create conditionally Mpp10p interaction-defective mutated Imp4ps. Because an essential gene encodes Imp4p, we screened for the subset of Mpp10p interaction-defective mutated Imp4ps that also conferred growth at the permissive temperature. Three different mutated Imp4ps were obtained that were interaction defective in the two-hybrid screen and conferred growth at the permissive but not at the non-permissive temperatures. Surprisingly, when tested by co-immunoprecipitation, Mpp10p±Imp4p *To whom correspondence should be addressed at Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, 333 Cedar Street, PO Box 208024, New Haven, CT 06530-8024, USA. Tel: +1 203 785 4618; Fax: +1 203 785 6404; Email: Nucleic Acids Research, Vol. 32 No. 4 ã Oxford University Press 2004; all rights reserved ABSTRACT Nucleic Acids Research, 2004, Vol. 32, No. 4 1405 interaction was not compromised at the non-permissive temperatures in the context of the SSU processome. However, pre-rRNA processing was defective at the nonpermissive temperatures, indicating a function for particular domains of Imp4p in speci®c pre-rRNA cleavage steps. injected monthly into guinea pigs and sera were tested for reactivity starting 3 months after the ®rst injection by western blots on puri®ed Imp4p and whole cell lysates. Anti-Mpp10p rabbit polyclonal antibodies were previously described (9). Anti-HA monoclonal antibodies were prepared from hybridoma cell line 12CA5. MATERIALS AND METHODS Mapping Mpp10p±Imp4p interacting domains Plasmids, strains and antibodies To map the interacting portions of Mpp10p with Imp4p, MPP10 and truncations of MPP10 were cloned into the plasmid encoding the GAL4 DNA binding domain (bait), pAS2-1. IMP4 was cloned into the plasmid with the GAL4 activation domain (prey), pACT-2. Strain MaV103 was used for the two-hybrid analysis (11). To map the interacting portions of Imp4p with Mpp10p, IMP4 and truncations of IMP4 were cloned into pAS2-1, and MPP10 was cloned into pACT-2. Strain pJ69-4A was used for the two-hybrid analysis (12). For the reverse two-hybrid screen, pAS2-1-imp4 was mutagenized and screened for interaction with the product of pACT-2-MPP10. Strain pJ69-4A was used for the screen. JA300, a tryptophan auxotropic Escherichia coli strain, was used to recover pAS2-1-imp4 plasmids (13). The imp4 alleles were expressed from the yeast constitutive plasmid p415GPD imp4 and shuf¯ed into the pGAL1::IMP4 (YPH259 Dimp4::HIS3) yeast strain. Polyclonal antibodies were raised against Imp4p puri®ed from E.coli expressed from the pET28 vector using the TALON kit (Clontech). At least 250 mg of puri®ed protein was pAS2-1-MPP10 (bait) was previously described (10) and consists of MPP10 encoding amino acids 1±498 fused to the GAL4 DNA binding domain. MPP10 truncations representing ami (...truncated)