Nucleolar Ribosome Assembly

Ribosomes are macromolecular machines that are responsible for production of every protein in a living cell. Yet we do not know the details about how these machines are formed. The ribosome consists of four RNA strands and roughly 80 proteins that associate with each other in the nucleolus and form pre-ribosomal complexes. Eukaryotes, in contrast to prokaryotes, need more than 200 non-ribosomal factors to assemble ribosomes. These associate with pre-ribosomal complexes at different stages as they travel from the nucleolus to the cytoplasm and are required for pre-rRNA processing. We do however lack knowledge about the molecular function of most of these factors and what enables pre-rRNA processing. Especially, information is missing about how non-ribosomal factors influence folding of the pre-rRNA and to what extent the pre-ribosomal complexes are restructured during their maturation.  This thesis aims to obtain a better understanding of the earliest events of ribosome assembly, namely those that take place in the nucleolus. This has been achieved by studying the essential protein Mrd1 by mutational analysis in the yeast Saccharomyces cerevisiae as well as by obtaining structural information of nucleolar pre-ribosomal complexes. Mrd1 has a modular structure consisting of multiple RNA binding domains (RBDs) that we find is conserved throughout eukarya. We show that an evolutionary conserved linker region of Mrd1 is crucial for function of the protein and likely forms an essential module together with adjacent RBDs. By obtaining structural information of pre-ribosomal complexes at different stages, we elucidate what structuring events occur in the nucleolus.  We uncover a direct role of Mrd1 in structuring the pre-rRNA in early pre-ribosomal complexes, which provides an explanation for why pre-rRNA cannot be processed in Mrd1 mutants.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p

Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2).

Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3

Schematic representation of Linker 2 and the analysed mutations.

<p>Linker 2 is positioned between RBD2 and RBD3. Numbers refer to amino acid residue positions in the <i>S</i>. <i>cerevisiae</i> Mrd1p. Linker definition and consensus sequence as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175506#pone.0175506.ref010" target="_blank">10</a>]. The most conserved part of Linker 2, between amino acid residues 439 and 524, contains four predicted α-helical regions (green boxes) and one β-strand (purple box). The <i>S</i>. <i>cerevisiae</i> Linker 2 amino acid sequence shared by homologues in other species is shown in bold. The conserved WN and AVK/R clusters are underlined. Single amino acid residue substitutions (W458A, K480E and K494E) are indicated together with three extensive mutations (Swap 1, Swap 2, Scrambled). In WNsubst and AVKsubst, the substitutions are shown in red and their locations are pointed out above the schematic representation of secondary structure predictions. In two additional mutants, 85 amino acid residues (curved line) were inserted, either between RBD2 and Linker 2 (5′-ins) or between Linker 2 and RBD3 (3′-ins).</p

Growth characteristics of Linker 2 mutants and expression of Mrd1 mutant proteins.

<p>A. Mutant <i>MRD1</i> genes were introduced into yeast cells, either as part of a plasmid (P_GAL<i>MRD1</i>+plasmid) or integrated into the genome (P_GAL<i>MRD1</i>+Genomic). In both cases, a WT <i>MRD1</i> gene was present in the genome, controlled by a <i>GAL1</i> promoter. The cells were diluted in steps (10 times for each step) and pipetted onto agar plates (from left to right), containing either galactose or glucose, followed by incubation at 30°C, 37°C or 16°C. Mutants are indicated to the left of each dilution series. The parental strain <i>mrd1-ΔL2</i>::<i>klURA3</i>, (FLY002, See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175506#pone.0175506.s003" target="_blank">S1 Table</a>), bearing a non-functional <i>MRD1</i> gene, served as a negative control for cell growth. B. Western blot analyses of the expression of Mrd1p in WT and mutant strains. Extracts from approximately 5x10⁵ cells were used. Ponceau staining demonstrated that approximately equal amounts of total proteins was loaded in each well (data not shown). The WT or mutant <i>MRD1</i> genes were either present in the genome or in a plasmid. The protein A part of the HTP tagged proteins was used for detection. WT Mrd1p is 101 kDa and its HTP tag is approximately 17 kDa. Migration of size marker proteins are shown (180, 130, 100, 70, kDa).</p

Sucrose gradient centrifugation analyses of Linker 2 mutants.

<p>Extracts from cells expressing WT Mrd1p or mutated Mrd1 proteins were centrifuged in a 10–50% a sucrose gradient and fractions were collected. Absorbance at 260 nm and positions of 25S and 18S rRNAs determined by Northern analyses were used to locate 80S ribosomes and 60S and 40S ribosomal subunits. Proteins in each fraction were analysed by Western blot. The tagged Mrd1 proteins were detected with anti-protein A antibody. Migration of size marker proteins is shown to the left (180, 130, 100, 70, kDa).</p

Sequence conservation characteristics of the individual RBDs.

<p>A. Consensus sequences of RBDs 1–6. The conserved residues are ordered according to frequency, with the most frequently occurring amino acid residue at the top. Rbm19 (human) residues are shown in bold. Secondary structure elements are derived as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g001" target="_blank">Fig. 1B</a>. B. Extent of conservation along each RBD. A window of five residues was slided along each of the six consensus RBDs, calculating the average presence of conserved residues (0–1, y-axis). This value is assigned to the central position of the window (solid line). The positions of α-helices are indicated in green and β-strands in red. C. Conserved residues in the 3D-structures of RBD2–6. Ribbon diagrams showing the 3D-structures of the human RBD2 (PDB identifier 2DGW) and the mouse RBD3–6 (PDB identifiers 1WHW, 1WHX, 2CPF and 2CPH, respectively), all in the same orientation, facing the β-sheet and with loops 1, 3 and 5 pointing downwards. Conserved residues are shown in red. In each RBD, the α1- and α2-helices as well as the β-strands (β1–β4) are labelled.</p

Dendrogram showing relationships between the RBDs.

<p>The six different RBDs form six clearly separated clusters, of which RBDs 1, 5 and 6 are most easily discernible. Each RBD is denoted with a two-letter code for species and a digit for the RBD number. Sequences are taken from: hs – <i>Homo sapiens</i> (Q9Y4C8), da – <i>Drosophila ananassae</i> (B3MYP1), ss – <i>Salpingoeca sp</i> (F2U536), mb – <i>Monosiga brevicollis</i> (A9USE7), cb – <i>Caenorhabditis briggsae</i> (A8WV73), bd – <i>Batrachochytrium dendrobatidis</i> (F4NSW1), dd – <i>Dictyostelium discoideum</i> (Q54PB2), tp – <i>Thalassiosira pseudonana</i> (B8BZC4), pi – <i>Phytophthora infestans</i> (D0NJ71), es – <i>Ectocarpus siliculosus</i> (D8LH81), at – <i>Arabidopsis thaliana</i> (F4JT92), ol – <i>Ostreococcus lucimarinus</i> (A4RVV1), tg – <i>Toxoplasma gondii</i> (B6KPW8), pm – <i>Perkinsus marinus</i> (C5KH14), sc – <i>Saccharomyces cerevisiae</i> (Q06106), pe – <i>Paramecium tetraurelia</i> (A0DWV5), ed – <i>Entamoeba dispar</i> (B0ECZ6), tc – <i>Trypanosoma cruzi</i> (E7KXH4), ng – <i>Naegleria gruberi</i> (D2V9G7), co – <i>Capsaspora owczarzaki</i> (E9C5E6), gl – <i>Giardia intestinalis</i> (A8BKE6). The number after each abbreviation indicates the RBD position.</p

Alignment of the microsporidia Rbm19/Mrd1 homologues to <i>S. cerevisiae</i> Mrd1.

<p>Alignment of RBDs 1, 3, 4, 6 and linker 3 of <i>S. cerevisiae</i> Mrd1 (denoted y) to <i>E. bieneusi</i> Mrd1 (denoted e). Identical residues (dark grey) or similar (light grey) between the two homologues are indicated. Secondary structure predictions are shown above the sequences. Positions present in the general consensus sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g005" target="_blank">Fig. 5A</a>) are underlined and asterisks indicate where 2 out of 3 of the microsporidia homologues (B7XJ60, C4V7E1 and E0S816) are conserved. Q8SRD9 was excluded due to high sequence similarity to E0S816 (>80% in the RBDs), in order to avoid bias.</p