The Noc-domain containing C-terminus of Noc4p mediates both formation of the Noc4p-Nop14p submodule and its incorporation into the SSU processome

Abstract

Noc1p, Noc3p and Noc4p are eukaryotic proteins which play essential roles in yeast ribosome biogenesis and contain a homologous stretch of about 45 aminoacids (Noc-domain) of unknown function. Yeast Noc4p is a component of the small ribosomal subunit (SSU) processome, can be isolated as a stable Noc4p-Nop14p SSU-processome submodule from yeast cells, and is required for nuclear steps of small ribosomal subunit rRNA maturation. We expressed a series of mutated alleles of NOC4 in yeast cells and analysed whether the corresponding protein variants support vegetative growth, interact with Nop14p, and are incorporated into the SSU-processome. The data reveal that the essential C-terminus of Noc4p which contains 237 aminoacids including the Noc-domain represents a protein-protein interaction module. It is required and sufficient for its association with Nop14p and several nuclear precursors of the small ribosomal subunit. The N-terminal Noc4-part seems to be targeted to pre-ribosomes via the C-terminus of Noc4p and plays there an essential role in SSU-processome function. Replacement of the Noc4p-Noc-domain by its homologues Noc1p-counterpart results in a hybrid Noc4p variant which fails to associate with Nop14p and pre-ribosomes. On the other hand, exchange of 6 amino acids in the Noc1-Noc-domain of this hybrid Noc4p protein is sufficient to restore its essential in vivo functions. These data suggest that Noc-domains of Noc1p and Noc4p share a common structural backbone in which diverging amino acids play crucial roles in mediating specific regulated interactions. Our analysis allows us to distinguish between different functions of certain domains within Noc4p and contribute to the understanding of how incorporation of Noc4p into ribosomal precursors is coupled to rRNA processing and maturation of the small ribosomal subunit.

Figure 1. Pathways of 18S rRNA precursor maturation in S. cerevisiae.

Three of the four rRNA-species found in mature ribosomes are generated from the 35S precursor RNA, which is processed in a series of defined endo- and exonucleolytic cleavages. The scheme shows an overview of 18S rRNA precursors found in S. cerevisiae and their suggested interrelationship. A main pre-18S rRNA processing pathway is thought to occur via early consecutive cleavages at sites A0, A1 and A2 leading to 20S pre-rRNA which is cleaved in the cytoplasm at site D to result in 18S rRNA. Alternative pre-18S rRNA processing pathways marked in the figure by letters a) and b) were proposed in , .

Figure 2. Deletion constructs of NOC4 and expression strategy.

(A) NOC4 was divided into eight domains according to their level of conservation with NOC4-homologues of other species. Deletion constructs were cloned in a vector under the control of the constitutive NOP1 promoter and were N-terminally fused to the ProteinA epitope tag. The gray box represents the Noc-domain (D7). (B) Strategy to perform in vivo interaction studies with non-viable deletion mutants of NOC4. Yeast strain Toy489 expressing wildtype NOC4 under the control of the glucose-repressible GAL1/10 promoter was transformed with plasmids coding for truncated Noc4p versions N-terminally fused with a Protein A tag under the control of the constitutive pNop1 promoter. Cells were cultivated in medium containing galactose as carbon source and then shifted for 16 h on glucose containing medium to shut off expression of wildtype NOC4 and to allow truncated Noc4p versions expressed to interact with pre-ribosomal components. ProtA-tagged Noc4p-variants were affinity purified using IgG-sepharose beads. (Note: all strains contained chromosomally tagged Nop14-HA).

Figure 3. Analysis of pre-ribosomal components associated with Noc4p-variants.

(A) Co-immunoprecipitation experiments of wild type cells and mutants expressing truncated Noc4p versions. Logarithmically growing yeast cells which express truncated versions of ProtA-Noc4p under the control of a constitutive promoter and wildtype Noc4p under the glucose repressible GAL1/10 promoter were shifted for 16 hours from galactose to glucose to shut off the expression of wildtype Noc4p. ProtA-Noc4p variants were affinitypurified and analyzed for association with Nop14p-HA by Western blotting and pre-18S rRNA and U3 snoRNA by Northern blotting. Noc4p-constructs were identified using a ProteinA antibody. Nop14p was detected by an antibody directed against its C-terminal HA epitope. Pre-rRNA and U3 snoRNA-species were detected using probes complementary to the ITS1 (D-A2) region of pre-rRNA's and U3-snoRNA, respectively. Signals in input lanes (In) correspond to 1% of cell extracts loaded onto IgG sepharose beads (IP lanes). (B) The C-terminal domain of Noc4p binds Nop14p when co-expressed in a heterologous baculovirus expression system. Equal numbers of SF21 insect cells were infected with recombinant MultiBac baculoviruses, carrying a HIS₆-NOP14 allele alone (control) or in combination with different Noc4 alleles (Flag-NOC4, Flag-noc4-dD1-D5, Flag-noc4-dD6-D8) and incubated for 48 hours. Flag-Noc4p variants were immunoprecipitated from the cell extracts using anti-flag M2 agarose beads and eluted with Flag-peptide. Aliquots of the eluates were analyzed for interactions between the Flag-Noc4p variants and HIS₆-Nop14p by Western blotting. (Co-)eluted proteins were detected using anti-Flag antibodies or antibodies directed against the HIS-tag, respectively. Equal volumes of eluates were loaded.

Figure 4. Depletion of Nop14p results in reduced Noc4p interaction with pre-ribosomes.

(A) Logarithmically growing yeast cells expressing NOP14 from a vector under the control of the glucose repressible GAL1/10 promoter were inoculated in YP-glucose containing medium for 16 h. Cell extracts before (lane 1) and after (lane 2) shift to glucose were prepared and equal amounts of protein were loaded onto IgG sepharose beads. Aliquots of the affinity purified fractions were analyzed by Western blotting for presence of Nop14p and by Northern blotting to determine association with pre-ribosomal RNAs (lanes 2 and 4). rRNA species were determined using probes complementary to the ITS1 (D-A2) and Noc4p was detected using a monoclonal Noc4p-antibody. (B) Quantitation of the Northern blot depicted in (A). The levels for each rRNA-species are normalized to the input. Signals in input lanes (In) correspond to 1% of cell extracts loaded onto IgG sepharose beads (IP lanes).

Figure 5. Exchange of Noc-domains within yeast Noc4p and determination of pathway-specific amino acids.

(A) The Noc-motif from Noc4p was substituted with the corresponding stretch of Noc1p, Noc3p or the putative Noc4p-homologue of H. sapiens, respectively. The hybrid alleles were cloned into a vector supporting their constitutive expression in yeast and the resulting plasmids (pTOKT87, pTOKT88, pTOKT89) were transformed into strain Toy489 expressing wildtype Noc4p under the control of the GAL1/10 promoter. Serial dilutions of yeast cells grown for 3 days on plates containing galactose or glucose as carbon source are shown. 1: NOC4 wildtype. 2: Noc4p-Noc-domain replaced by Noc1-Noc-domain. 3: Noc4p-Noc-domain replaced by Noc3-Noc-domain. 4: Noc4p-Noc-domain replaced by the corresponding stretch of human Noc4p. (B) Cells expressing Noc4p under the control of the pGAL1/10 promoter (Toy489) and Noc4p-hybrids N-terminally decorated by an ProteinA-tag under the constitutive pNop1 promoter were shifted for 16 h from galactose containing medium to glucose containing medium to shut down expression of wild type NOC4. Cell extracts were prepared and equal amounts of protein were loaded onto IgG sepharose beads. Aliquots of the precipitates were analyzed for association of Protein tagged Noc4p variants with Nop14p (Western blot) and to determine copurification with pre-ribosomal RNAs and U3 snoRNA (Northern blot). Nop14p was detected by an antibody directed against its C-terminally fused HA epitope, Noc4p-hybrid constructs via its N-terminal fused ProteinA tag. Pre-rRNA species were determined using probes complementary to the ITS1 (D-A2) region of pre-rRNA, U3 snoRNA was detected with a complementary oligo probe. Signals in input lanes (In) correspond to 1% of cell extracts loaded onto IgG sepharose beads (IP lanes). (C) Sequence comparison of Noc-domains analysed in this study. Lanes 1–3 shows the sequences of the Noc-domains of Noc1p, Noc3p and Noc4p from S. cerevisiae, respectively, lane 4 shows the corresponding sequence region of the putative human Noc4p homologue. Exchange of the six encircled residues in the Noc1p sequence with the corresponding aminoacids in Noc4p sequence resulted in a hybrid Noc4p - Noc1p allele which is able to complement the essential functions of of yeast NOC4.