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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 Rpl22l1 expression impairs cellular growth.(A) Lysates isolated Rpl22+/+ and Rpl22−/− 3T9 cells treated with or without doxycycline were collected to assess levels of Rpl22 or Rpl22L1 by Western blot analysis. GAPDH was used as a loading control. (B) Growth of Rpl22+/+ and Rpl22−/− 3T9 cells was compared. Levels of Rpl22l1 were analyzed by Western blot analysis to confirm that the Rpl22l1-shRNA knocked down levels of Rpl22l1 in doxycycline-treated Rpl22+/+ (C) and Rpl22−/− (E) 3T9 cells transduced with the shRNA construct. Growth of Rpl22+/+ (D) and Rpl22−/− (F) 3T9 cells transduced with each shRNA construct was determined. Results are representative of 2 independent experiments with error bars representative of ±SD. Statistical significance is indicated (*, p<0.05 compared to untreated control).
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pgen-1003708-g006: Acute knockdown of Rpl22l1 expression impairs cellular growth.(A) Lysates isolated Rpl22+/+ and Rpl22−/− 3T9 cells treated with or without doxycycline were collected to assess levels of Rpl22 or Rpl22L1 by Western blot analysis. GAPDH was used as a loading control. (B) Growth of Rpl22+/+ and Rpl22−/− 3T9 cells was compared. Levels of Rpl22l1 were analyzed by Western blot analysis to confirm that the Rpl22l1-shRNA knocked down levels of Rpl22l1 in doxycycline-treated Rpl22+/+ (C) and Rpl22−/− (E) 3T9 cells transduced with the shRNA construct. Growth of Rpl22+/+ (D) and Rpl22−/− (F) 3T9 cells transduced with each shRNA construct was determined. Results are representative of 2 independent experiments with error bars representative of ±SD. Statistical significance is indicated (*, p<0.05 compared to untreated control).

Mentions: In yeast, ribosomal protein paralogs are thought to functionally compensate for one another, each incorporating into the ribosome in the absence of the other. Deletion of both yeast RPL22 paralogs results in viable, but slow growing cells [37]. Recently Rpl22+/− or Rpl22−/− primary mouse embryonic fibroblasts (MEFs) were found to grow faster and display increased transformation potential relative to MEFs isolated from Rpl22+/+ littermates [45] To further evaluate the effect of Rpl22 and Rpl22Ll1 expression on growth rates, Rpl22+/+ or Rpl22−/− 3T9 fibroblasts were transduced with one of 2 different tet-on shRNA lentivirus constructs (shRNA#1- and shRNA#2-Rpl22l1) that target Rpl22l1 mRNA to acutely knock-down its expression. Western blot analysis confirmed that Rpl22l1 protein levels were elevated in Rpl22−/− 3T9 fibroblasts (Figure 6A). No significant difference was observed in the growth rates of Rpl22+/+ or Rpl22−/− 3T9 fibroblasts (Figure 6B). In doxycycline-treated Rpl22+/+ or Rpl22−/− 3T9 fibroblasts expressing the shRNA constructs, Rpl22l1 protein levels were confirmed to be reduced by western blot analysis (Figure 6C, E). Knockdown of Rpl22l1 significantly reduced growth rates of Rpl22+/+ fibroblasts and greatly impaired that of Rpl22−/− 3T9 fibroblasts (Figure 6D, F and S6), indicating that cells lacking both paralogs have severe growth defects. In contrast, acute knockdown of Rpl22 in Rpl22+/+ 3T9 fibroblasts resulted in no change in the rate of proliferation (Figure S7). In summary, these cell culture studies indicate that expression of at least one paralog of Rpl22 is required for normal growth and suggests that Rpl22l1 may also affect cell proliferation by a mechanism independent of Rpl22.


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 Rpl22l1 expression impairs cellular growth.(A) Lysates isolated Rpl22+/+ and Rpl22−/− 3T9 cells treated with or without doxycycline were collected to assess levels of Rpl22 or Rpl22L1 by Western blot analysis. GAPDH was used as a loading control. (B) Growth of Rpl22+/+ and Rpl22−/− 3T9 cells was compared. Levels of Rpl22l1 were analyzed by Western blot analysis to confirm that the Rpl22l1-shRNA knocked down levels of Rpl22l1 in doxycycline-treated Rpl22+/+ (C) and Rpl22−/− (E) 3T9 cells transduced with the shRNA construct. Growth of Rpl22+/+ (D) and Rpl22−/− (F) 3T9 cells transduced with each shRNA construct was determined. Results are representative of 2 independent experiments with error bars representative of ±SD. Statistical significance is indicated (*, p<0.05 compared to untreated control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750023&req=5

pgen-1003708-g006: Acute knockdown of Rpl22l1 expression impairs cellular growth.(A) Lysates isolated Rpl22+/+ and Rpl22−/− 3T9 cells treated with or without doxycycline were collected to assess levels of Rpl22 or Rpl22L1 by Western blot analysis. GAPDH was used as a loading control. (B) Growth of Rpl22+/+ and Rpl22−/− 3T9 cells was compared. Levels of Rpl22l1 were analyzed by Western blot analysis to confirm that the Rpl22l1-shRNA knocked down levels of Rpl22l1 in doxycycline-treated Rpl22+/+ (C) and Rpl22−/− (E) 3T9 cells transduced with the shRNA construct. Growth of Rpl22+/+ (D) and Rpl22−/− (F) 3T9 cells transduced with each shRNA construct was determined. Results are representative of 2 independent experiments with error bars representative of ±SD. Statistical significance is indicated (*, p<0.05 compared to untreated control).
Mentions: In yeast, ribosomal protein paralogs are thought to functionally compensate for one another, each incorporating into the ribosome in the absence of the other. Deletion of both yeast RPL22 paralogs results in viable, but slow growing cells [37]. Recently Rpl22+/− or Rpl22−/− primary mouse embryonic fibroblasts (MEFs) were found to grow faster and display increased transformation potential relative to MEFs isolated from Rpl22+/+ littermates [45] To further evaluate the effect of Rpl22 and Rpl22Ll1 expression on growth rates, Rpl22+/+ or Rpl22−/− 3T9 fibroblasts were transduced with one of 2 different tet-on shRNA lentivirus constructs (shRNA#1- and shRNA#2-Rpl22l1) that target Rpl22l1 mRNA to acutely knock-down its expression. Western blot analysis confirmed that Rpl22l1 protein levels were elevated in Rpl22−/− 3T9 fibroblasts (Figure 6A). No significant difference was observed in the growth rates of Rpl22+/+ or Rpl22−/− 3T9 fibroblasts (Figure 6B). In doxycycline-treated Rpl22+/+ or Rpl22−/− 3T9 fibroblasts expressing the shRNA constructs, Rpl22l1 protein levels were confirmed to be reduced by western blot analysis (Figure 6C, E). Knockdown of Rpl22l1 significantly reduced growth rates of Rpl22+/+ fibroblasts and greatly impaired that of Rpl22−/− 3T9 fibroblasts (Figure 6D, F and S6), indicating that cells lacking both paralogs have severe growth defects. In contrast, acute knockdown of Rpl22 in Rpl22+/+ 3T9 fibroblasts resulted in no change in the rate of proliferation (Figure S7). In summary, these cell culture studies indicate that expression of at least one paralog of Rpl22 is required for normal growth and suggests that Rpl22l1 may also affect cell proliferation by a mechanism independent of Rpl22.

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