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Rational extension of the ribosome biogenesis pathway using network-guided genetics.

Li Z, Lee I, Moradi E, Hung NJ, Johnson AW, Marcotte EM - PLoS Biol. (2009)

Bottom Line: Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes.We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs.These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, USA.

ABSTRACT
Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

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Co-sedimentation of candidate proteins with ribosomal subunits was independently verified using mass spectrometry.(A) Schematic overview of the experimental design. (B) Hierarchical clustering of abundance profiles of 1,023 proteins (row) identified from fractions (columns) of the sucrose density gradient of wild-type yeast cells. Four distinct clusters are enriched (p<10−8; [83]) for r-proteins, translation-initiation factors and 40S biogenesis factors, metabolic enzymes, and 60S biogenesis factors. Representative profiles are plotted for r-proteins (C), 40S biogenesis factors (D), metabolic enzymes (E), and 60S biogenesis factors (F). (G–J) show profiles for several ribosome biogenesis candidates. Abundance in (C–J) is provided as the frequency of spectral counts (×10,000) of each protein in each fraction; abundance in (B) is further row-normalized.
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pbio-1000213-g004: Co-sedimentation of candidate proteins with ribosomal subunits was independently verified using mass spectrometry.(A) Schematic overview of the experimental design. (B) Hierarchical clustering of abundance profiles of 1,023 proteins (row) identified from fractions (columns) of the sucrose density gradient of wild-type yeast cells. Four distinct clusters are enriched (p<10−8; [83]) for r-proteins, translation-initiation factors and 40S biogenesis factors, metabolic enzymes, and 60S biogenesis factors. Representative profiles are plotted for r-proteins (C), 40S biogenesis factors (D), metabolic enzymes (E), and 60S biogenesis factors (F). (G–J) show profiles for several ribosome biogenesis candidates. Abundance in (C–J) is provided as the frequency of spectral counts (×10,000) of each protein in each fraction; abundance in (B) is further row-normalized.

Mentions: In order to assay protein co-sedimentation with pre-ribosomes in a tag-independent fashion, we employed a shotgun-style tandem mass-spectrometry (MS/MS) approach [36]. Proteins in each of 14 fractions from a sucrose density gradient separation of the whole-cell lysate from wild-type yeast were identified by mass spectrometry and quantified using MS/MS spectral counts (Figure 4A), which measured the proportion of the total observed MS/MS spectra that were associated with each given protein [37]. Using an approach shown to quantitatively map protein separation profiles in complex samples [38], the distribution of each protein along the density gradient was derived from the normalized abundance profiles obtained across the set of mass-spectrometry analyses (Figure 4A). We identified, on the basis of their sedimentation profiles, a total of 1,023 unique proteins (Table S3) that were clustered into four major groups (Figure 4B; sedimentation profiles of representatives for each group are shown in Figure 4C–4F). Most r-proteins distributed in the high-density fractions corresponding to the polysomes (Figures 4B, 4C), and many translation-initiation factors and 40S biogenesis factors were clustered together and sedimented in the 40S fractions (Figures 4B, 4D). One group primarily distributing in the low-density fractions was highly enriched for metabolic enzymes (Figures 4B, 4E). Finally, many 60S subunit biogenesis factors sedimented in the 60S fractions (Figures 4B, 4F). As controls, the distributions of marker proteins for each of these groups (Eno1p, Tsr1p, Lsg1p, Rps3p, and Rpl8ap) were determined and were found to be consistent with their sedimentation patterns as measured by immunoblot (Figure 3A, Figure 4C–4F), which supports this mass-spectrometry-based approach to measuring the sedimentation pattern for each protein.


Rational extension of the ribosome biogenesis pathway using network-guided genetics.

Li Z, Lee I, Moradi E, Hung NJ, Johnson AW, Marcotte EM - PLoS Biol. (2009)

Co-sedimentation of candidate proteins with ribosomal subunits was independently verified using mass spectrometry.(A) Schematic overview of the experimental design. (B) Hierarchical clustering of abundance profiles of 1,023 proteins (row) identified from fractions (columns) of the sucrose density gradient of wild-type yeast cells. Four distinct clusters are enriched (p<10−8; [83]) for r-proteins, translation-initiation factors and 40S biogenesis factors, metabolic enzymes, and 60S biogenesis factors. Representative profiles are plotted for r-proteins (C), 40S biogenesis factors (D), metabolic enzymes (E), and 60S biogenesis factors (F). (G–J) show profiles for several ribosome biogenesis candidates. Abundance in (C–J) is provided as the frequency of spectral counts (×10,000) of each protein in each fraction; abundance in (B) is further row-normalized.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749941&req=5

pbio-1000213-g004: Co-sedimentation of candidate proteins with ribosomal subunits was independently verified using mass spectrometry.(A) Schematic overview of the experimental design. (B) Hierarchical clustering of abundance profiles of 1,023 proteins (row) identified from fractions (columns) of the sucrose density gradient of wild-type yeast cells. Four distinct clusters are enriched (p<10−8; [83]) for r-proteins, translation-initiation factors and 40S biogenesis factors, metabolic enzymes, and 60S biogenesis factors. Representative profiles are plotted for r-proteins (C), 40S biogenesis factors (D), metabolic enzymes (E), and 60S biogenesis factors (F). (G–J) show profiles for several ribosome biogenesis candidates. Abundance in (C–J) is provided as the frequency of spectral counts (×10,000) of each protein in each fraction; abundance in (B) is further row-normalized.
Mentions: In order to assay protein co-sedimentation with pre-ribosomes in a tag-independent fashion, we employed a shotgun-style tandem mass-spectrometry (MS/MS) approach [36]. Proteins in each of 14 fractions from a sucrose density gradient separation of the whole-cell lysate from wild-type yeast were identified by mass spectrometry and quantified using MS/MS spectral counts (Figure 4A), which measured the proportion of the total observed MS/MS spectra that were associated with each given protein [37]. Using an approach shown to quantitatively map protein separation profiles in complex samples [38], the distribution of each protein along the density gradient was derived from the normalized abundance profiles obtained across the set of mass-spectrometry analyses (Figure 4A). We identified, on the basis of their sedimentation profiles, a total of 1,023 unique proteins (Table S3) that were clustered into four major groups (Figure 4B; sedimentation profiles of representatives for each group are shown in Figure 4C–4F). Most r-proteins distributed in the high-density fractions corresponding to the polysomes (Figures 4B, 4C), and many translation-initiation factors and 40S biogenesis factors were clustered together and sedimented in the 40S fractions (Figures 4B, 4D). One group primarily distributing in the low-density fractions was highly enriched for metabolic enzymes (Figures 4B, 4E). Finally, many 60S subunit biogenesis factors sedimented in the 60S fractions (Figures 4B, 4F). As controls, the distributions of marker proteins for each of these groups (Eno1p, Tsr1p, Lsg1p, Rps3p, and Rpl8ap) were determined and were found to be consistent with their sedimentation patterns as measured by immunoblot (Figure 3A, Figure 4C–4F), which supports this mass-spectrometry-based approach to measuring the sedimentation pattern for each protein.

Bottom Line: Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes.We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs.These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, USA.

ABSTRACT
Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics-an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies-to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes-most with human orthologs-to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.

Show MeSH
Related in: MedlinePlus