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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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The U24 snoRNA is responsible for the 60S biogenesis defect observed in an asc1Δ mutant.Polysome profile of wild-type strain with two control plasmids was shown in (A). The asc1Δ mutant with two control plasmids showed the 60S biogenesis defect (B), and this defect was recovered by full length intron-containing ASC1 gene (C) or intron snoRNA U24 of ASC1 (D), but not by the coded protein of ASC1, deleted of its intron (E). When the intron snoRNA U24 of ASC1 was put back into the asc1Δ mutant expressing the coded protein of ASC1, the polysome profile recovered to wild-type (F). All strains were cultured at 37°C. pRS416 and pRS413-ACT are the control plasmids; pRS416-ASC1 carries full length ASC1 with both intron and exon; pRS413-ACT/U24 carries the intron sequence of ASC1; and pRS416-ASC1ORF carries the sequence of protein coding region of ASC1. Peaks corresponding to 40S and 60S ribosomal subunits and 80S mono-ribosomes in the polysome profiles were labeled. Gray arrows indicate the halfmer polysomes.
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pbio-1000213-g006: The U24 snoRNA is responsible for the 60S biogenesis defect observed in an asc1Δ mutant.Polysome profile of wild-type strain with two control plasmids was shown in (A). The asc1Δ mutant with two control plasmids showed the 60S biogenesis defect (B), and this defect was recovered by full length intron-containing ASC1 gene (C) or intron snoRNA U24 of ASC1 (D), but not by the coded protein of ASC1, deleted of its intron (E). When the intron snoRNA U24 of ASC1 was put back into the asc1Δ mutant expressing the coded protein of ASC1, the polysome profile recovered to wild-type (F). All strains were cultured at 37°C. pRS416 and pRS413-ACT are the control plasmids; pRS416-ASC1 carries full length ASC1 with both intron and exon; pRS413-ACT/U24 carries the intron sequence of ASC1; and pRS416-ASC1ORF carries the sequence of protein coding region of ASC1. Peaks corresponding to 40S and 60S ribosomal subunits and 80S mono-ribosomes in the polysome profiles were labeled. Gray arrows indicate the halfmer polysomes.

Mentions: Among the mutants we found to exhibit 27S processing defects, the gene ASC1 was particularly notable: ASC1 contains an intron that encodes U24 C/D box small nucleolar RNA required for 2′-O-methylation of 25S at C1437, C1449, and C1450 [53], whereas Asc1 protein has been shown to be a component of the 40S subunit [54]. We observed reductions in 27S, 20S, and 25S upon deletion of both the intron and exons of ASC1 when cultured at 37°C (Figure 5E), which is consistent with reduced levels of 60S subunits observed in the polysome profile (Figure 2C). In order to determine whether the intron or protein conferred the observed defect, we tested complementation of the asc1Δ with each: expression of U24 in asc1Δ partially suppressed 60S biogenesis defects observed in the polysome profile analysis, whereas expression of Asc1 protein did not alleviate the defects (Figure 6), which indicates the importance of U24 instead of Asc1p in 60S biogenesis. Ribosomal RNA modifications by snoRNAs have been known for a long time, but their exact physiological roles are generally unclear. Recently, 20 C/D box snoRNAs were shown to phenotypically affect ribosomes [55], and here we demonstrate that rRNA modifications by the intron-encoded snoRNA U24 affect the formation of 60S subunits, demonstrating the importance of an individual snoRNA in ribosome biogenesis.


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)

The U24 snoRNA is responsible for the 60S biogenesis defect observed in an asc1Δ mutant.Polysome profile of wild-type strain with two control plasmids was shown in (A). The asc1Δ mutant with two control plasmids showed the 60S biogenesis defect (B), and this defect was recovered by full length intron-containing ASC1 gene (C) or intron snoRNA U24 of ASC1 (D), but not by the coded protein of ASC1, deleted of its intron (E). When the intron snoRNA U24 of ASC1 was put back into the asc1Δ mutant expressing the coded protein of ASC1, the polysome profile recovered to wild-type (F). All strains were cultured at 37°C. pRS416 and pRS413-ACT are the control plasmids; pRS416-ASC1 carries full length ASC1 with both intron and exon; pRS413-ACT/U24 carries the intron sequence of ASC1; and pRS416-ASC1ORF carries the sequence of protein coding region of ASC1. Peaks corresponding to 40S and 60S ribosomal subunits and 80S mono-ribosomes in the polysome profiles were labeled. Gray arrows indicate the halfmer polysomes.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000213-g006: The U24 snoRNA is responsible for the 60S biogenesis defect observed in an asc1Δ mutant.Polysome profile of wild-type strain with two control plasmids was shown in (A). The asc1Δ mutant with two control plasmids showed the 60S biogenesis defect (B), and this defect was recovered by full length intron-containing ASC1 gene (C) or intron snoRNA U24 of ASC1 (D), but not by the coded protein of ASC1, deleted of its intron (E). When the intron snoRNA U24 of ASC1 was put back into the asc1Δ mutant expressing the coded protein of ASC1, the polysome profile recovered to wild-type (F). All strains were cultured at 37°C. pRS416 and pRS413-ACT are the control plasmids; pRS416-ASC1 carries full length ASC1 with both intron and exon; pRS413-ACT/U24 carries the intron sequence of ASC1; and pRS416-ASC1ORF carries the sequence of protein coding region of ASC1. Peaks corresponding to 40S and 60S ribosomal subunits and 80S mono-ribosomes in the polysome profiles were labeled. Gray arrows indicate the halfmer polysomes.
Mentions: Among the mutants we found to exhibit 27S processing defects, the gene ASC1 was particularly notable: ASC1 contains an intron that encodes U24 C/D box small nucleolar RNA required for 2′-O-methylation of 25S at C1437, C1449, and C1450 [53], whereas Asc1 protein has been shown to be a component of the 40S subunit [54]. We observed reductions in 27S, 20S, and 25S upon deletion of both the intron and exons of ASC1 when cultured at 37°C (Figure 5E), which is consistent with reduced levels of 60S subunits observed in the polysome profile (Figure 2C). In order to determine whether the intron or protein conferred the observed defect, we tested complementation of the asc1Δ with each: expression of U24 in asc1Δ partially suppressed 60S biogenesis defects observed in the polysome profile analysis, whereas expression of Asc1 protein did not alleviate the defects (Figure 6), which indicates the importance of U24 instead of Asc1p in 60S biogenesis. Ribosomal RNA modifications by snoRNAs have been known for a long time, but their exact physiological roles are generally unclear. Recently, 20 C/D box snoRNAs were shown to phenotypically affect ribosomes [55], and here we demonstrate that rRNA modifications by the intron-encoded snoRNA U24 affect the formation of 60S subunits, demonstrating the importance of an individual snoRNA in ribosome biogenesis.

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