The Noc-Domain Containing C-Terminus of Noc4p Mediates Both Formation of the Noc4p-Nop14p Submodule and Its Incorporation into the SSU Processome

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

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks

Characterisation of the Rrp5p/Noc1p/Noc2p protein complex and its function in ribosome biogenesis of Saccharomyces cerevisiae

Eukaryotic ribosome biogenesis is a very complex process that includes synthesis of the structural components (ribosomal RNAs (rRNAs) and proteins (r-proteins)), processing and folding of rRNA precursors, as well as assembly of the r-proteins onto the rRNA. Ribosome biogenesis starts with the transcription of the genes encoding the rRNAs (rDNA) in the nucleolus by RNA polymerase I and III, and includes transport of pre-ribosomal particles (pre-ribosomes) through the nucleus and export into the cytoplasm, where the final maturation steps occur. In addition to the structural components, these processes require the function of ~75 small nucleolar RNAs and of more than 150 non-ribosomal proteins termed biogenesis factors, which transiently interact with different pre-ribosomes. It could be shown that several subsets of biogenesis factors form protein modules, which are supposed to constitute building blocks of pre-ribosomes and/or to function together in ribosome biogenesis. In this work, a protein complex consisting of the proteins Rrp5p, Noc1p and Noc2p from Saccharomyces cerevisiae could be reconstituted from heterologously expressed proteins. Noc1p and Noc2p are biogenesis factors of the large ribosomal subunit (LSU), whereas Rrp5p is required for maturation of both the large and the small ribosomal subunit (SSU). Analyses of pairwise interactions between the proteins, as well as negative stain electron microscopy of the purified complex provided further insights into architectural and structural features of the Rrp5p/Noc1p/Noc2p biogenesis factor module. Ex vivo purifications of the module components and analyses of co-purified RNAs and proteins indicated that the Rrp5p/Noc1p/Noc2p module is predominantly associated with the first specific pre-LSU particles. In addition, Rrp5p, Noc1p and Noc2p showed association with early, common ribosomal precursor particles, which are formed before the pathways leading to the small and the large ribosomal subunit are separated. Furthermore, the module components co-purified specific regions of rDNA chromatin from cells treated with crosslinking reagents, and Rrp5p and Noc1p were identified as components of chromatin transcribed by RNA polymerase I. Accordingly, the Rrp5p/Noc1p/Noc2p module appeared to be associated with nascent rRNA precursor transcripts, providing further evidence that the module is recruited very early in ribosome biogenesis. Individual inactivation or depletion of Rrp5p, Noc1p or Noc2p in vivo resulted in severely decreased levels of LSU specific pre-rRNA species and the appearance of aberrant pre-rRNA fragments. In addition, analyses of truncated noc1 alleles indicated that impaired interactions of Noc1p with Noc2p, Rrp5p or pre-rRNA result in similar pre-rRNA processing phenotypes, suggesting that in absence of the Rrp5p/Noc1p/Noc2p module pre-ribosomes are destabilised and pre-rRNAs are prone to degradation. Furthermore, in vivo depletion of one module component and subsequent analyses of the association of the respective non-depleted proteins with pre-rRNA indicated a mutually independent binding of Rrp5p and Noc1p/Noc2p to pre-ribosomes. Accordingly, the module most probably has several binding sites on pre-ribosomal particles. In summary, the results presented here suggest that formation of the Rrp5p/Noc1p/Noc2p module plays a role in the structural organisation of early LSU precursor particles and thereby contributes to their stability, possibly by preventing inappropriate access of endo- and exonucleases to pre-rRNA. Besides, potential mechanisms of the Noc1p/Noc2p independent function of Rrp5p in SSU biogenesis, and a model for the recruitment of the Rrp5p/Noc1p/Noc2p module to pre-ribosomes are discussed. Future studies will be required to determine the structure and architecture of this biogenesis factor module in detail. Furthermore, analyses of the RNA binding and folding activities of the module components, and of the impact of the module on the recruitment of r-proteins and/or other biogenesis factors to early pre-ribosomes will help to understand the precise molecular function of the Rrp5p/Noc1p/Noc2p module in ribosome biogenesis. As all three proteins have homologues in higher eukaryotes, it will be interesting to investigate if formation and function of this module are conserved in evolution

Local Tertiary Structure Probing of Ribonucleoprotein Particles by Nuclease Fusion Proteins

Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed

In vitro reconstitution of yeast tUTP/UTP A and UTP B subcomplexes provides new insights into their modular architecture.

Eukaryotic ribosome biogenesis is a multistep process involving more than 150 biogenesis factors, which interact transiently with pre-ribosomal particles to promote their maturation. Some of these auxiliary proteins have been isolated in complexes found separate from the ribosomal environment. Among them, are 3 large UTP subcomplexes containing 6 or 7 protein subunits which are involved in the early steps of ribosome biogenesis. The composition of the UTP subcomplexes and the network of binary interactions between protein subunits have been analyzed previously. To obtain further insights into the structural and biochemical properties of UTP subcomplexes, we established a heterologous expression system to allow reconstitution of the yeast tUTP/UTP A and UTP B subcomplexes from their candidate subunits. The results of a series of reconstitution experiments involving different combinations of protein subunits are in good agreement with most of the previously observed binary interactions. Moreover, in combination with additional biochemical analyses, several stable building blocks of the UTP subcomplexes were identified. Based on these findings, we present a refined model of the tUTP/UTP A and UTP B architecture

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed

Yeast UTP B subcomplex reconstitution in insect cells.

<p>All selected UTP B components were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991 or K1992. The protein content of the indicated bands was identified by MS and are indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. (<b>A</b>) Lysates of 2×10⁸ cells infected with K1991were used for two-step affinity purification. Pwp2-TAP was used as the bait protein in the first affinity purification step with IgG-coupled Sepharose resin, and Pwp2-containing components were eluted with TEV protease (Lane 1). Utp6-HA-containing components were purified from 90% of the first elution sample using anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of the eluate (5% each) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. (<b>B</b>) Lysates of 2×10⁸ cells infected with K1992 were used for two-step affinity purification. Utp12-FLAG was purified with anti-FLAG affinity matrix and eluted with the FLAG peptide during the first affinity purification step (Lane 1). A 90% aliquot of the eluted material was used to purify Utp6-HA-containing components with anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of both eluates (5%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. (<b>C</b>) Lysates of 8×10⁷ SF21 cells infected with bacmid K1991 were cleared by the low-speed centrifugation described in the normal protocol (N samples), and half was further cleared by ultracentrifugation (200000×<i>g</i>, 1 h, 4°C, U samples). Pwp2-TAP-containing components were purified from both lysates using IgG-coupled Sepharose resin and eluted with TEV protease. A 10% aliquot of the eluted material was analyzed on a 4-12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed with MS. (<b>D</b>) Pwp2-TAP-containing components were purified from lysates of 4×10⁷ infected cells (K1991) using IgG-coupled Sepharose resin and TEV elution. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), flow through from the first purification (FT, 0,03%), the eluate from the affinity column (E, 10%), and the fractions from the gel filtration column (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and WB with antibodies against CBP (middle panel) or HA (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification by MS analysis of the corresponding protein is indicated.</p

Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.

<p>Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.</p

Identification of different UTP B building blocks.

<p>Tagged proteins were purified from cell extracts containing different UTP B components in one or two step affinity purifications. Correct identification by MS analysis of the corresponding protein is indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. Expression of the tagged proteins is indicated as +: untagged protein expressed; T:TAP-tagged; F: FLAG-tagged; *: bait protein. (<b>A</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K1987, K2134, K2135, K2136, K1991 and K1978. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease (Lanes 1–6) or with anti-FLAG affinity beads and elution with FLAG peptide (Lane 7). Samples of the elution were analyzed with SDS-PAGE and MS analysis. (<b>B</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K2134, K2136, K2138 and K2139. Expression of the tagged proteins is indicated. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with anti-FLAG affinity matrix and eluted with the FLAG peptide. Samples of the elution were analyzed with SDS-PAGE and MS analysis. Note that a band compatible with the size of Utp4-TAP is observed in Lane 3 but was not possible to characterize by MS analysis. (<b>C</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991, K2134, K2135, K2136, K2137 and K1987. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease. Aliquots of the elution (upper panel) or of the corresponding cell lysate (lower panel) were analyzed by WB with anti-HA antibody. The corresponding co-expressed proteins are indicated at the top of the figure.</p