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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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Rpl22 directly binds Rpl22l1 mRNA to regulate its expression levels.(A) In the absence of Rpl22, Rpl22l1 mRNA levels are more stable in the presence of Actinomycin D. Rpl22+/+ or Rpl22−/− 3T9 cells were treated with Actinomycin D (1 µM final concentration) and total RNA was harvested at the time points shown. Levels of Rpl22l1 mRNA were quantitated by qRT-PCR. Results are the average ± SEM of 3 independent experiments and the statistical significance indicated is (*, p<0.01, compared to Rpl22+/+ untreated; ** p<0.001, compared to Rpl22+/+ at each time point). (B) M-fold analysis [54] of zRpl22l1 mRNA reveals the presence of a consensus Rpl22 RNA-binding motif. In green are the residues deleted to remove the hairpin (zRpl22l1Δhp). In blue are the residues known to be essential for Rpl22 binding. (C) Autoradiogram of ribonuclease protection assay reveals Rpl22 protein binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. 32P labeled EBER1 (positive control), EBER 2 (negative control), zRpl22l1 or zRpl22l1Δhp RNAs were incubated in the absence or presence of GST-Rpl22 (41.7 kDa), GST (27 kDa) or m88, a GST-Rpl22 RNA binding mutant (41.6 kDa), as indicated, then UV-cross-linked, digested with RNase A, and run on a SDS protein gel. GST-Rpl22 was detected, hence, bound to EBER1 and zRpl22l1 RNAs but not Rpl22l1Δhp RNA, indicating Rpl22 binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. Numbers indicate molecular weight protein ladder in kDa.
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pgen-1003708-g004: Rpl22 directly binds Rpl22l1 mRNA to regulate its expression levels.(A) In the absence of Rpl22, Rpl22l1 mRNA levels are more stable in the presence of Actinomycin D. Rpl22+/+ or Rpl22−/− 3T9 cells were treated with Actinomycin D (1 µM final concentration) and total RNA was harvested at the time points shown. Levels of Rpl22l1 mRNA were quantitated by qRT-PCR. Results are the average ± SEM of 3 independent experiments and the statistical significance indicated is (*, p<0.01, compared to Rpl22+/+ untreated; ** p<0.001, compared to Rpl22+/+ at each time point). (B) M-fold analysis [54] of zRpl22l1 mRNA reveals the presence of a consensus Rpl22 RNA-binding motif. In green are the residues deleted to remove the hairpin (zRpl22l1Δhp). In blue are the residues known to be essential for Rpl22 binding. (C) Autoradiogram of ribonuclease protection assay reveals Rpl22 protein binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. 32P labeled EBER1 (positive control), EBER 2 (negative control), zRpl22l1 or zRpl22l1Δhp RNAs were incubated in the absence or presence of GST-Rpl22 (41.7 kDa), GST (27 kDa) or m88, a GST-Rpl22 RNA binding mutant (41.6 kDa), as indicated, then UV-cross-linked, digested with RNase A, and run on a SDS protein gel. GST-Rpl22 was detected, hence, bound to EBER1 and zRpl22l1 RNAs but not Rpl22l1Δhp RNA, indicating Rpl22 binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. Numbers indicate molecular weight protein ladder in kDa.

Mentions: Collectively, these data suggest that Rpl22 is regulating expression of Rpl22l1 but the mechanism leading to the increased expression is unknown. To determine whether Rpl22 affects the stability of Rpl22l1 mRNA, cultures of 3T9 cells were treated with actinomycin D, which blocks transcription by all three eukaryotic polymerases [52]. After actinomycin D treatment, the levels of Rpl22l1 mRNA in Rpl22+/+ 3T9 cells decreased significantly (p<0.01) relative to the untreated control, while in Rpl22−/− 3T9 cells Rpl22l1 levels were maintained and the rate of decay was reduced (Figure 4A). These results suggest that Rpl22 affects the stability of Rpl22l1 mRNA.


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)

Rpl22 directly binds Rpl22l1 mRNA to regulate its expression levels.(A) In the absence of Rpl22, Rpl22l1 mRNA levels are more stable in the presence of Actinomycin D. Rpl22+/+ or Rpl22−/− 3T9 cells were treated with Actinomycin D (1 µM final concentration) and total RNA was harvested at the time points shown. Levels of Rpl22l1 mRNA were quantitated by qRT-PCR. Results are the average ± SEM of 3 independent experiments and the statistical significance indicated is (*, p<0.01, compared to Rpl22+/+ untreated; ** p<0.001, compared to Rpl22+/+ at each time point). (B) M-fold analysis [54] of zRpl22l1 mRNA reveals the presence of a consensus Rpl22 RNA-binding motif. In green are the residues deleted to remove the hairpin (zRpl22l1Δhp). In blue are the residues known to be essential for Rpl22 binding. (C) Autoradiogram of ribonuclease protection assay reveals Rpl22 protein binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. 32P labeled EBER1 (positive control), EBER 2 (negative control), zRpl22l1 or zRpl22l1Δhp RNAs were incubated in the absence or presence of GST-Rpl22 (41.7 kDa), GST (27 kDa) or m88, a GST-Rpl22 RNA binding mutant (41.6 kDa), as indicated, then UV-cross-linked, digested with RNase A, and run on a SDS protein gel. GST-Rpl22 was detected, hence, bound to EBER1 and zRpl22l1 RNAs but not Rpl22l1Δhp RNA, indicating Rpl22 binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. Numbers indicate molecular weight protein ladder in kDa.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003708-g004: Rpl22 directly binds Rpl22l1 mRNA to regulate its expression levels.(A) In the absence of Rpl22, Rpl22l1 mRNA levels are more stable in the presence of Actinomycin D. Rpl22+/+ or Rpl22−/− 3T9 cells were treated with Actinomycin D (1 µM final concentration) and total RNA was harvested at the time points shown. Levels of Rpl22l1 mRNA were quantitated by qRT-PCR. Results are the average ± SEM of 3 independent experiments and the statistical significance indicated is (*, p<0.01, compared to Rpl22+/+ untreated; ** p<0.001, compared to Rpl22+/+ at each time point). (B) M-fold analysis [54] of zRpl22l1 mRNA reveals the presence of a consensus Rpl22 RNA-binding motif. In green are the residues deleted to remove the hairpin (zRpl22l1Δhp). In blue are the residues known to be essential for Rpl22 binding. (C) Autoradiogram of ribonuclease protection assay reveals Rpl22 protein binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. 32P labeled EBER1 (positive control), EBER 2 (negative control), zRpl22l1 or zRpl22l1Δhp RNAs were incubated in the absence or presence of GST-Rpl22 (41.7 kDa), GST (27 kDa) or m88, a GST-Rpl22 RNA binding mutant (41.6 kDa), as indicated, then UV-cross-linked, digested with RNase A, and run on a SDS protein gel. GST-Rpl22 was detected, hence, bound to EBER1 and zRpl22l1 RNAs but not Rpl22l1Δhp RNA, indicating Rpl22 binds to Rpl22l1 mRNA and this binding is abrogated upon removal of the hairpin. Numbers indicate molecular weight protein ladder in kDa.
Mentions: Collectively, these data suggest that Rpl22 is regulating expression of Rpl22l1 but the mechanism leading to the increased expression is unknown. To determine whether Rpl22 affects the stability of Rpl22l1 mRNA, cultures of 3T9 cells were treated with actinomycin D, which blocks transcription by all three eukaryotic polymerases [52]. After actinomycin D treatment, the levels of Rpl22l1 mRNA in Rpl22+/+ 3T9 cells decreased significantly (p<0.01) relative to the untreated control, while in Rpl22−/− 3T9 cells Rpl22l1 levels were maintained and the rate of decay was reduced (Figure 4A). These results suggest that Rpl22 affects the stability of Rpl22l1 mRNA.

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