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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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Regulation of Rpl22l1 mRNA expression is mediated by a hairpin structure.(A) Schematic representation of the biosensor quantification assay. (B–D) Stereoimages of zebrafish embryos illustrate that co-injection of zRpl22 repressed fluorescence derived from an EGFP-Rpl22l1 fusion protein upon injecting mRNA for both and assessing fluorescence at 6 hours post fertilization. Rpl22, Rpl22l1 or mutated Rpl22l1 (Rpl22l1mt) coding sequence was fused to EGFP mRNA and co-injected with mCherry mRNA (injection control) along with the corresponding inhibitor mRNAs (zRpl22 or zRpl22l1) into 1-cell stage zebrafish embryos. (F) Schematic representation of the experimental procedure. A zRpl22l1-150h-EGFP heterologous reporter mRNA, containing the minimal sequence identified by mFOLD to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (G) Rpl22 mRNA or (I) Rpl22-Morpholino (Rpl22-MO) into 1-cell stage zebrafish embryos. (E, H, J) At 10 hpf, the relative fluorescence intensity was calculated and normalized to control injections (n = 3, each group). Data are shown as mean ± standard deviation (s.d.).
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pgen-1003708-g005: Regulation of Rpl22l1 mRNA expression is mediated by a hairpin structure.(A) Schematic representation of the biosensor quantification assay. (B–D) Stereoimages of zebrafish embryos illustrate that co-injection of zRpl22 repressed fluorescence derived from an EGFP-Rpl22l1 fusion protein upon injecting mRNA for both and assessing fluorescence at 6 hours post fertilization. Rpl22, Rpl22l1 or mutated Rpl22l1 (Rpl22l1mt) coding sequence was fused to EGFP mRNA and co-injected with mCherry mRNA (injection control) along with the corresponding inhibitor mRNAs (zRpl22 or zRpl22l1) into 1-cell stage zebrafish embryos. (F) Schematic representation of the experimental procedure. A zRpl22l1-150h-EGFP heterologous reporter mRNA, containing the minimal sequence identified by mFOLD to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (G) Rpl22 mRNA or (I) Rpl22-Morpholino (Rpl22-MO) into 1-cell stage zebrafish embryos. (E, H, J) At 10 hpf, the relative fluorescence intensity was calculated and normalized to control injections (n = 3, each group). Data are shown as mean ± standard deviation (s.d.).

Mentions: To determine if Rpl22 directly regulates expression of Rpl22l1, we employed a biosensor quantification assay using GFP as a fluorescent indicator of effects on expression. Zebrafish embryos were microinjected with mRNAs EGFP-zRpl22, EGFP-Rpl22l1 or a mutant form of Rpl22l1 in which the Rpl22l1 hairpin was modified (EGFP-zRpl22L1mt) in combination with constructs expressing zRpl22 or zRpl22l1. mCherry mRNA was co-injected to allow for quantification of the relative fluorescence intensity. Co-injection of zRpl22 with EGFP-Rpl22l1 led to a significant decreased fluorescence relative to those embryos co-injected with zRpl22 and EGFP-Rpl22l1mt (Figure 5A–E), suggesting that the hairpin structure within Rpl22l1 mRNA is necessary for Rpl22 to directly regulate its expression. Next, to assess if the presence of the hairpin structure within the Rpl22l1 mRNA is sufficient to regulate expression, the hairpin sequence from zRpl22l1 mRNA was fused to EGFP and evaluated in the biosensor quantification assay. The heterologous reporter mRNA, zRpl22l1-150h-EGFP, containing the minimal sequence identified by M-fold to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (Figure 5G) zRpl22 mRNA or (Figure 5I) Rpl22-Morpholino (Rpl22-MO) into zebrafish embryos. Rpl22 repressed the expression of zRpl22l1-150h-EGFP reporter, while knockdown Rpl22 can increase the expression of reporter, suggesting the hairpin structure identified in zRpl22l1 mRNA is sufficient to regulate mRNA abundance.


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)

Regulation of Rpl22l1 mRNA expression is mediated by a hairpin structure.(A) Schematic representation of the biosensor quantification assay. (B–D) Stereoimages of zebrafish embryos illustrate that co-injection of zRpl22 repressed fluorescence derived from an EGFP-Rpl22l1 fusion protein upon injecting mRNA for both and assessing fluorescence at 6 hours post fertilization. Rpl22, Rpl22l1 or mutated Rpl22l1 (Rpl22l1mt) coding sequence was fused to EGFP mRNA and co-injected with mCherry mRNA (injection control) along with the corresponding inhibitor mRNAs (zRpl22 or zRpl22l1) into 1-cell stage zebrafish embryos. (F) Schematic representation of the experimental procedure. A zRpl22l1-150h-EGFP heterologous reporter mRNA, containing the minimal sequence identified by mFOLD to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (G) Rpl22 mRNA or (I) Rpl22-Morpholino (Rpl22-MO) into 1-cell stage zebrafish embryos. (E, H, J) At 10 hpf, the relative fluorescence intensity was calculated and normalized to control injections (n = 3, each group). Data are shown as mean ± standard deviation (s.d.).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750023&req=5

pgen-1003708-g005: Regulation of Rpl22l1 mRNA expression is mediated by a hairpin structure.(A) Schematic representation of the biosensor quantification assay. (B–D) Stereoimages of zebrafish embryos illustrate that co-injection of zRpl22 repressed fluorescence derived from an EGFP-Rpl22l1 fusion protein upon injecting mRNA for both and assessing fluorescence at 6 hours post fertilization. Rpl22, Rpl22l1 or mutated Rpl22l1 (Rpl22l1mt) coding sequence was fused to EGFP mRNA and co-injected with mCherry mRNA (injection control) along with the corresponding inhibitor mRNAs (zRpl22 or zRpl22l1) into 1-cell stage zebrafish embryos. (F) Schematic representation of the experimental procedure. A zRpl22l1-150h-EGFP heterologous reporter mRNA, containing the minimal sequence identified by mFOLD to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (G) Rpl22 mRNA or (I) Rpl22-Morpholino (Rpl22-MO) into 1-cell stage zebrafish embryos. (E, H, J) At 10 hpf, the relative fluorescence intensity was calculated and normalized to control injections (n = 3, each group). Data are shown as mean ± standard deviation (s.d.).
Mentions: To determine if Rpl22 directly regulates expression of Rpl22l1, we employed a biosensor quantification assay using GFP as a fluorescent indicator of effects on expression. Zebrafish embryos were microinjected with mRNAs EGFP-zRpl22, EGFP-Rpl22l1 or a mutant form of Rpl22l1 in which the Rpl22l1 hairpin was modified (EGFP-zRpl22L1mt) in combination with constructs expressing zRpl22 or zRpl22l1. mCherry mRNA was co-injected to allow for quantification of the relative fluorescence intensity. Co-injection of zRpl22 with EGFP-Rpl22l1 led to a significant decreased fluorescence relative to those embryos co-injected with zRpl22 and EGFP-Rpl22l1mt (Figure 5A–E), suggesting that the hairpin structure within Rpl22l1 mRNA is necessary for Rpl22 to directly regulate its expression. Next, to assess if the presence of the hairpin structure within the Rpl22l1 mRNA is sufficient to regulate expression, the hairpin sequence from zRpl22l1 mRNA was fused to EGFP and evaluated in the biosensor quantification assay. The heterologous reporter mRNA, zRpl22l1-150h-EGFP, containing the minimal sequence identified by M-fold to form the hairpin structure, was co-injected with mCherry mRNA (injection control) and (Figure 5G) zRpl22 mRNA or (Figure 5I) Rpl22-Morpholino (Rpl22-MO) into zebrafish embryos. Rpl22 repressed the expression of zRpl22l1-150h-EGFP reporter, while knockdown Rpl22 can increase the expression of reporter, suggesting the hairpin structure identified in zRpl22l1 mRNA is sufficient to regulate mRNA abundance.

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