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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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Acute knockdown of Rpl22 enhances Rpl22l1 expression.3T9 cells were transduced with doxycycline-inducible shRNA lentiviral constructs directed at Rpl22 (shRNA 1 and shRNA 2) or a non-specific (ns) shRNA construct and treated with doxycycline for 3 days to induce shRNA expression. NS, shRNA 1, or shRNA 2 expressing cells were analyzed for relative Rpl22 and Rpl22l1 mRNA levels by qRT-PCR (A) or protein expression by Western blot analysis (B). Results are the average ± SEM of 4 independent experiments (A) or representative of 3 independent experiments (B). Statistical significance is indicated (*; p<0.001 compared to untreated control).
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pgen-1003708-g003: Acute knockdown of Rpl22 enhances Rpl22l1 expression.3T9 cells were transduced with doxycycline-inducible shRNA lentiviral constructs directed at Rpl22 (shRNA 1 and shRNA 2) or a non-specific (ns) shRNA construct and treated with doxycycline for 3 days to induce shRNA expression. NS, shRNA 1, or shRNA 2 expressing cells were analyzed for relative Rpl22 and Rpl22l1 mRNA levels by qRT-PCR (A) or protein expression by Western blot analysis (B). Results are the average ± SEM of 4 independent experiments (A) or representative of 3 independent experiments (B). Statistical significance is indicated (*; p<0.001 compared to untreated control).

Mentions: Why does expression of Rpl22l1 increase in mouse tissues lacking Rpl22? We considered two potential explanations: (1) Rpl22 directly regulates Rpl22l1 expression, or (2) compensation occurs during development in Rpl22−/− mice. To distinguish between these two possibilities, Rpl22 was acutely knocked down in 3T9 fibroblasts using a lentiviral-mediated inducible knockdown system that allows doxycycline-inducible regulation of Rpl22 and changes in Rpl22l1 expression were examined. 3T9 cells were transduced with 2 different tet-on shRNA lentivirus constructs (shRNA 1 and shRNA 2) that target Rpl22 mRNA or a nonspecific control construct. Following 3 days of doxycycline treatment, Rpl22l1 mRNA expression is enhanced 1.8 fold in 3T9 cells with reduced Rpl22 expression (Figure 3A). Western blot analysis confirmed that Rpl22l1 protein levels were elevated by the knockdown of Rpl22, while expression of other RPs, such as Rpl7 remained unchanged (Figure 3B). These results confirm that Rpl22 negatively regulates the expression of Rpl22l1 acutely and raise the possibility that Rpl22-mediated regulation of Rpl22l1 is an active process with biological significance.


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)

Acute knockdown of Rpl22 enhances Rpl22l1 expression.3T9 cells were transduced with doxycycline-inducible shRNA lentiviral constructs directed at Rpl22 (shRNA 1 and shRNA 2) or a non-specific (ns) shRNA construct and treated with doxycycline for 3 days to induce shRNA expression. NS, shRNA 1, or shRNA 2 expressing cells were analyzed for relative Rpl22 and Rpl22l1 mRNA levels by qRT-PCR (A) or protein expression by Western blot analysis (B). Results are the average ± SEM of 4 independent experiments (A) or representative of 3 independent experiments (B). Statistical significance is indicated (*; p<0.001 compared to untreated control).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750023&req=5

pgen-1003708-g003: Acute knockdown of Rpl22 enhances Rpl22l1 expression.3T9 cells were transduced with doxycycline-inducible shRNA lentiviral constructs directed at Rpl22 (shRNA 1 and shRNA 2) or a non-specific (ns) shRNA construct and treated with doxycycline for 3 days to induce shRNA expression. NS, shRNA 1, or shRNA 2 expressing cells were analyzed for relative Rpl22 and Rpl22l1 mRNA levels by qRT-PCR (A) or protein expression by Western blot analysis (B). Results are the average ± SEM of 4 independent experiments (A) or representative of 3 independent experiments (B). Statistical significance is indicated (*; p<0.001 compared to untreated control).
Mentions: Why does expression of Rpl22l1 increase in mouse tissues lacking Rpl22? We considered two potential explanations: (1) Rpl22 directly regulates Rpl22l1 expression, or (2) compensation occurs during development in Rpl22−/− mice. To distinguish between these two possibilities, Rpl22 was acutely knocked down in 3T9 fibroblasts using a lentiviral-mediated inducible knockdown system that allows doxycycline-inducible regulation of Rpl22 and changes in Rpl22l1 expression were examined. 3T9 cells were transduced with 2 different tet-on shRNA lentivirus constructs (shRNA 1 and shRNA 2) that target Rpl22 mRNA or a nonspecific control construct. Following 3 days of doxycycline treatment, Rpl22l1 mRNA expression is enhanced 1.8 fold in 3T9 cells with reduced Rpl22 expression (Figure 3A). Western blot analysis confirmed that Rpl22l1 protein levels were elevated by the knockdown of Rpl22, while expression of other RPs, such as Rpl7 remained unchanged (Figure 3B). These results confirm that Rpl22 negatively regulates the expression of Rpl22l1 acutely and raise the possibility that Rpl22-mediated regulation of Rpl22l1 is an active process with biological significance.

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