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The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1.

O'Leary MN, Schreiber KH, Zhang Y, Duc AC, Rao S, Hale JS, Academia EC, Shah SR, Morton JF, Holstein CA, Martin DB, Kaeberlein M, Ladiges WC, Fink PJ, Mackay VL, Wiest DL, Kennedy BK - PLoS Genet. (2013)

Bottom Line: Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast.Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression.We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22(-/-) mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22(-/-) mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

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Both mouse Rpl22 and Rpl22l1 proteins can be incorporated into ribosomes.Liver tissue was isolated from Rpl22+/+ (A, C) and Rpl22−/− (B, D) mice then, after sedimentation of the lysates on sucrose gradients, fractions were collected and loaded onto an SDS-page gel for western blot analysis (C and D, respectively). Images are representative of 3 independent experiments. Multiple Reaction Monitoring Mass spectrometry (MRM-MS) analysis was performed on free 60S subunits and 80S monosomes from actively translating polysomes. Liver lysates from Rpl22+/+ and Rpl22−/− mice were subjected to a brief treatment with low amounts of RNase A to degrade mRNA between ribosomes in polysomes and release the ribosomes as 80S monomers. After inhibiting the RNase with KCl and heparin, the samples were fractionated on 10–30% sucrose gradients containing 800 mM KCl to disrupt any “nonproductive couples” of 40S and 60S subunits [79]. (E, F) Representative gradient profiles for Rpl22+/+ and Rpl22−/−samples, respectively. Fraction 6 was used to isolate 60S subunits for MRM-MS, while fraction 7 was used to isolate 80S monosomes. Summation of the integrated MRM peak areas for all transitions from all observed peptides for Rpl22 (G) and Rpl22l1 (H) proteins yielded the total MRM peak areas plotted for each of the four tested samples (WT60S, WT80S, KO60S, KO80S), indicating the relative amounts of Rpl22 and Rpl22l1 in these samples. The height of each bar represents the average of the three technical replicates performed for the given sample, and each error bar represents +/−1 standard deviation. p-values>3E-3 in both cases by paired student's t-test. Rpl22 peptides: AGNLGGGVVTIER; ITVTSEVPFSK; YFQINQDEEEEEDED. Rpl22l1 peptides: TGNLGNVVHIER; ITVVSEK.
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pgen-1003708-g002: Both mouse Rpl22 and Rpl22l1 proteins can be incorporated into ribosomes.Liver tissue was isolated from Rpl22+/+ (A, C) and Rpl22−/− (B, D) mice then, after sedimentation of the lysates on sucrose gradients, fractions were collected and loaded onto an SDS-page gel for western blot analysis (C and D, respectively). Images are representative of 3 independent experiments. Multiple Reaction Monitoring Mass spectrometry (MRM-MS) analysis was performed on free 60S subunits and 80S monosomes from actively translating polysomes. Liver lysates from Rpl22+/+ and Rpl22−/− mice were subjected to a brief treatment with low amounts of RNase A to degrade mRNA between ribosomes in polysomes and release the ribosomes as 80S monomers. After inhibiting the RNase with KCl and heparin, the samples were fractionated on 10–30% sucrose gradients containing 800 mM KCl to disrupt any “nonproductive couples” of 40S and 60S subunits [79]. (E, F) Representative gradient profiles for Rpl22+/+ and Rpl22−/−samples, respectively. Fraction 6 was used to isolate 60S subunits for MRM-MS, while fraction 7 was used to isolate 80S monosomes. Summation of the integrated MRM peak areas for all transitions from all observed peptides for Rpl22 (G) and Rpl22l1 (H) proteins yielded the total MRM peak areas plotted for each of the four tested samples (WT60S, WT80S, KO60S, KO80S), indicating the relative amounts of Rpl22 and Rpl22l1 in these samples. The height of each bar represents the average of the three technical replicates performed for the given sample, and each error bar represents +/−1 standard deviation. p-values>3E-3 in both cases by paired student's t-test. Rpl22 peptides: AGNLGGGVVTIER; ITVTSEVPFSK; YFQINQDEEEEEDED. Rpl22l1 peptides: TGNLGNVVHIER; ITVVSEK.

Mentions: To determine if Rpl22l1 is incorporated into actively translating ribosomes, liver tissue was isolated from Rpl22−/− mice and their littermate controls followed by sedimentation of the lysates on sucrose gradients. Fractions collected from the gradients, were subsequently loaded onto an SDS-page gel for western blot analysis. In Rpl22−/− samples, Rpl22l1 is present in fractions containing 60S ribosome subunits and polysomes, suggesting that it is incorporated into free ribosome subunits and ribosomes actively translating mRNA in the absence of Rpl22(Figure 2). Rpl7, a RP that is incorporated into the large subunit of the ribosome, is present in the fractions containing 60S ribosome subunits and polysomes in both Rpl22+/+ and Rpl22−/− samples. Rpl22 and Rpl22l1, but not Rpl7, were detected in fractions 1 and 2, representative of the free, non-ribosomal lysate (Figure 2C, D), consistent with the hypothesis that these RPs exist in states within the cell both associated with the ribosome and independent of the ribosome. Additionally, while Rpl22l1 levels are relatively evenly detected in 60S containing fractions of the polysome profile (Figure 2D, fractions 3–7) Rpl22 is detected at higher levels in the fractions containing free 60S subunits and messages loaded with fewer ribosomes (Figure 2C, fractions 3–5 vs 6–7).


The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1.

O'Leary MN, Schreiber KH, Zhang Y, Duc AC, Rao S, Hale JS, Academia EC, Shah SR, Morton JF, Holstein CA, Martin DB, Kaeberlein M, Ladiges WC, Fink PJ, Mackay VL, Wiest DL, Kennedy BK - PLoS Genet. (2013)

Both mouse Rpl22 and Rpl22l1 proteins can be incorporated into ribosomes.Liver tissue was isolated from Rpl22+/+ (A, C) and Rpl22−/− (B, D) mice then, after sedimentation of the lysates on sucrose gradients, fractions were collected and loaded onto an SDS-page gel for western blot analysis (C and D, respectively). Images are representative of 3 independent experiments. Multiple Reaction Monitoring Mass spectrometry (MRM-MS) analysis was performed on free 60S subunits and 80S monosomes from actively translating polysomes. Liver lysates from Rpl22+/+ and Rpl22−/− mice were subjected to a brief treatment with low amounts of RNase A to degrade mRNA between ribosomes in polysomes and release the ribosomes as 80S monomers. After inhibiting the RNase with KCl and heparin, the samples were fractionated on 10–30% sucrose gradients containing 800 mM KCl to disrupt any “nonproductive couples” of 40S and 60S subunits [79]. (E, F) Representative gradient profiles for Rpl22+/+ and Rpl22−/−samples, respectively. Fraction 6 was used to isolate 60S subunits for MRM-MS, while fraction 7 was used to isolate 80S monosomes. Summation of the integrated MRM peak areas for all transitions from all observed peptides for Rpl22 (G) and Rpl22l1 (H) proteins yielded the total MRM peak areas plotted for each of the four tested samples (WT60S, WT80S, KO60S, KO80S), indicating the relative amounts of Rpl22 and Rpl22l1 in these samples. The height of each bar represents the average of the three technical replicates performed for the given sample, and each error bar represents +/−1 standard deviation. p-values>3E-3 in both cases by paired student's t-test. Rpl22 peptides: AGNLGGGVVTIER; ITVTSEVPFSK; YFQINQDEEEEEDED. Rpl22l1 peptides: TGNLGNVVHIER; ITVVSEK.
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Related In: Results  -  Collection

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pgen-1003708-g002: Both mouse Rpl22 and Rpl22l1 proteins can be incorporated into ribosomes.Liver tissue was isolated from Rpl22+/+ (A, C) and Rpl22−/− (B, D) mice then, after sedimentation of the lysates on sucrose gradients, fractions were collected and loaded onto an SDS-page gel for western blot analysis (C and D, respectively). Images are representative of 3 independent experiments. Multiple Reaction Monitoring Mass spectrometry (MRM-MS) analysis was performed on free 60S subunits and 80S monosomes from actively translating polysomes. Liver lysates from Rpl22+/+ and Rpl22−/− mice were subjected to a brief treatment with low amounts of RNase A to degrade mRNA between ribosomes in polysomes and release the ribosomes as 80S monomers. After inhibiting the RNase with KCl and heparin, the samples were fractionated on 10–30% sucrose gradients containing 800 mM KCl to disrupt any “nonproductive couples” of 40S and 60S subunits [79]. (E, F) Representative gradient profiles for Rpl22+/+ and Rpl22−/−samples, respectively. Fraction 6 was used to isolate 60S subunits for MRM-MS, while fraction 7 was used to isolate 80S monosomes. Summation of the integrated MRM peak areas for all transitions from all observed peptides for Rpl22 (G) and Rpl22l1 (H) proteins yielded the total MRM peak areas plotted for each of the four tested samples (WT60S, WT80S, KO60S, KO80S), indicating the relative amounts of Rpl22 and Rpl22l1 in these samples. The height of each bar represents the average of the three technical replicates performed for the given sample, and each error bar represents +/−1 standard deviation. p-values>3E-3 in both cases by paired student's t-test. Rpl22 peptides: AGNLGGGVVTIER; ITVTSEVPFSK; YFQINQDEEEEEDED. Rpl22l1 peptides: TGNLGNVVHIER; ITVVSEK.
Mentions: To determine if Rpl22l1 is incorporated into actively translating ribosomes, liver tissue was isolated from Rpl22−/− mice and their littermate controls followed by sedimentation of the lysates on sucrose gradients. Fractions collected from the gradients, were subsequently loaded onto an SDS-page gel for western blot analysis. In Rpl22−/− samples, Rpl22l1 is present in fractions containing 60S ribosome subunits and polysomes, suggesting that it is incorporated into free ribosome subunits and ribosomes actively translating mRNA in the absence of Rpl22(Figure 2). Rpl7, a RP that is incorporated into the large subunit of the ribosome, is present in the fractions containing 60S ribosome subunits and polysomes in both Rpl22+/+ and Rpl22−/− samples. Rpl22 and Rpl22l1, but not Rpl7, were detected in fractions 1 and 2, representative of the free, non-ribosomal lysate (Figure 2C, D), consistent with the hypothesis that these RPs exist in states within the cell both associated with the ribosome and independent of the ribosome. Additionally, while Rpl22l1 levels are relatively evenly detected in 60S containing fractions of the polysome profile (Figure 2D, fractions 3–7) Rpl22 is detected at higher levels in the fractions containing free 60S subunits and messages loaded with fewer ribosomes (Figure 2C, fractions 3–5 vs 6–7).

Bottom Line: Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast.Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression.We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22(-/-) mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22(-/-) mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

Show MeSH
Related in: MedlinePlus