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Differential Stoichiometry among Core Ribosomal Proteins.

Slavov N, Semrau S, Airoldi E, Budnik B, van Oudenaarden A - Cell Rep (2015)

Bottom Line: Testing such variability requires direct and precise quantification of RPs.Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes.Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Northeastern University, Boston, MA 02115, USA; Department of Statistics and FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: nslavov@alum.mit.edu.

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The Stoichiometry among Core RPs in Mouse Ribosomes Depends on the Number of Ribosomes per mRNA(A) Velocity sedimentation in sucrose gradients allow separating ribosomes that are free or bound to a single mRNA (monosomes, depicted in black) from multiple ribosomes bound to a single mRNA (polysomes, depicted in blue). The absorbance at 254 nm reflects RNA levels, mostly ribosomal RNA. The vertical dashed lines indicate the boundaries of the collected fractions. Fractions are labeled at the top with numbers reflecting the number of ribosomes per mRNA.(B) Replicates MS measurements of the monosomes (A and B) indicate reproducible estimates for RP enrichment in polysomes.(C and D) Some RPs are enriched in monosomes (C) and others in polysomes (D). The relative levels of each RP are quantified as the median levels of its unique peptides, and the probability that the RP levels do not change across the quantified fractions is computed from ANOVA (indicated at the top). The distributions of levels of all unique peptides from trypsin (left panels) and from lys-C (right panels) digestions are juxtaposed as boxplots to depict the consistency of the estimates across proteases, different peptides, and experiments.For each fraction, the mean intensity of all RP peptides was normalized to 1. On each box, the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points.See also Figures S1 and S2.
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fig1: The Stoichiometry among Core RPs in Mouse Ribosomes Depends on the Number of Ribosomes per mRNA(A) Velocity sedimentation in sucrose gradients allow separating ribosomes that are free or bound to a single mRNA (monosomes, depicted in black) from multiple ribosomes bound to a single mRNA (polysomes, depicted in blue). The absorbance at 254 nm reflects RNA levels, mostly ribosomal RNA. The vertical dashed lines indicate the boundaries of the collected fractions. Fractions are labeled at the top with numbers reflecting the number of ribosomes per mRNA.(B) Replicates MS measurements of the monosomes (A and B) indicate reproducible estimates for RP enrichment in polysomes.(C and D) Some RPs are enriched in monosomes (C) and others in polysomes (D). The relative levels of each RP are quantified as the median levels of its unique peptides, and the probability that the RP levels do not change across the quantified fractions is computed from ANOVA (indicated at the top). The distributions of levels of all unique peptides from trypsin (left panels) and from lys-C (right panels) digestions are juxtaposed as boxplots to depict the consistency of the estimates across proteases, different peptides, and experiments.For each fraction, the mean intensity of all RP peptides was normalized to 1. On each box, the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points.See also Figures S1 and S2.

Mentions: To explore whether the stoichiometry among core RPs can vary, we first isolated monosomes and polysomes from exponentially growing mouse embryonic stem cells (ESC), doubling every 9 hr, Figure S1A. The ESC ribosomes were isolated by velocity sedimentation in sucrose gradients (Figure 1A); see Experimental Procedures. To confirm that the prominent monosomal peak is reflective of ESC biology and not of poor ribosome fractionation, we also fractionated the ribosomes of neuroprogenitor cells derived from the ESC. Despite growing three times slower (doubling time 29 hr) than the ESC, the neuroprogenitor cells have a larger fraction of their ribosomes in polysomal complexes, Figure S1B. This observation confirms earlier findings by Sampath et al. (2008), and thus further bolsters the conclusion that a low polysome-to-monosomes ratio is characteristic of ESC.


Differential Stoichiometry among Core Ribosomal Proteins.

Slavov N, Semrau S, Airoldi E, Budnik B, van Oudenaarden A - Cell Rep (2015)

The Stoichiometry among Core RPs in Mouse Ribosomes Depends on the Number of Ribosomes per mRNA(A) Velocity sedimentation in sucrose gradients allow separating ribosomes that are free or bound to a single mRNA (monosomes, depicted in black) from multiple ribosomes bound to a single mRNA (polysomes, depicted in blue). The absorbance at 254 nm reflects RNA levels, mostly ribosomal RNA. The vertical dashed lines indicate the boundaries of the collected fractions. Fractions are labeled at the top with numbers reflecting the number of ribosomes per mRNA.(B) Replicates MS measurements of the monosomes (A and B) indicate reproducible estimates for RP enrichment in polysomes.(C and D) Some RPs are enriched in monosomes (C) and others in polysomes (D). The relative levels of each RP are quantified as the median levels of its unique peptides, and the probability that the RP levels do not change across the quantified fractions is computed from ANOVA (indicated at the top). The distributions of levels of all unique peptides from trypsin (left panels) and from lys-C (right panels) digestions are juxtaposed as boxplots to depict the consistency of the estimates across proteases, different peptides, and experiments.For each fraction, the mean intensity of all RP peptides was normalized to 1. On each box, the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points.See also Figures S1 and S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4644233&req=5

fig1: The Stoichiometry among Core RPs in Mouse Ribosomes Depends on the Number of Ribosomes per mRNA(A) Velocity sedimentation in sucrose gradients allow separating ribosomes that are free or bound to a single mRNA (monosomes, depicted in black) from multiple ribosomes bound to a single mRNA (polysomes, depicted in blue). The absorbance at 254 nm reflects RNA levels, mostly ribosomal RNA. The vertical dashed lines indicate the boundaries of the collected fractions. Fractions are labeled at the top with numbers reflecting the number of ribosomes per mRNA.(B) Replicates MS measurements of the monosomes (A and B) indicate reproducible estimates for RP enrichment in polysomes.(C and D) Some RPs are enriched in monosomes (C) and others in polysomes (D). The relative levels of each RP are quantified as the median levels of its unique peptides, and the probability that the RP levels do not change across the quantified fractions is computed from ANOVA (indicated at the top). The distributions of levels of all unique peptides from trypsin (left panels) and from lys-C (right panels) digestions are juxtaposed as boxplots to depict the consistency of the estimates across proteases, different peptides, and experiments.For each fraction, the mean intensity of all RP peptides was normalized to 1. On each box, the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points.See also Figures S1 and S2.
Mentions: To explore whether the stoichiometry among core RPs can vary, we first isolated monosomes and polysomes from exponentially growing mouse embryonic stem cells (ESC), doubling every 9 hr, Figure S1A. The ESC ribosomes were isolated by velocity sedimentation in sucrose gradients (Figure 1A); see Experimental Procedures. To confirm that the prominent monosomal peak is reflective of ESC biology and not of poor ribosome fractionation, we also fractionated the ribosomes of neuroprogenitor cells derived from the ESC. Despite growing three times slower (doubling time 29 hr) than the ESC, the neuroprogenitor cells have a larger fraction of their ribosomes in polysomal complexes, Figure S1B. This observation confirms earlier findings by Sampath et al. (2008), and thus further bolsters the conclusion that a low polysome-to-monosomes ratio is characteristic of ESC.

Bottom Line: Testing such variability requires direct and precise quantification of RPs.Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes.Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Northeastern University, Boston, MA 02115, USA; Department of Statistics and FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: nslavov@alum.mit.edu.

Show MeSH
Related in: MedlinePlus