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A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

Yeo JH, Skinner JP, Bird MJ, Formosa LE, Zhang JG, Kluck RM, Belz GT, Chong MM - PLoS ONE (2015)

Bottom Line: This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts.We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome.These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia; St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.

ABSTRACT
We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

No MeSH data available.


Knockdown of Mrpl44 affects the expression of mtDNA-encoded genes.Protein and RNA were extracted from NIH3T3 knocked down (ORF) for Mrpl44 or expressing a control shRNA. (A) RNA expression of mitochondrial genes was analyzed by quantitative RT-PCR. Expression was normalized to β-actin. Statistical analysis was performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005). The mean +/- SEM of 3 independent experiments is shown. (B) Western blotting for the OXPHOS proteins COX IV and ND6, mitoribosome proteins Mrpl44, Mrpl11 and Mrps15. The mitochondrial protein Grp75 was analysed as a loading control. A representative of three independent experiments is shown.
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pone.0134326.g003: Knockdown of Mrpl44 affects the expression of mtDNA-encoded genes.Protein and RNA were extracted from NIH3T3 knocked down (ORF) for Mrpl44 or expressing a control shRNA. (A) RNA expression of mitochondrial genes was analyzed by quantitative RT-PCR. Expression was normalized to β-actin. Statistical analysis was performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005). The mean +/- SEM of 3 independent experiments is shown. (B) Western blotting for the OXPHOS proteins COX IV and ND6, mitoribosome proteins Mrpl44, Mrpl11 and Mrps15. The mitochondrial protein Grp75 was analysed as a loading control. A representative of three independent experiments is shown.

Mentions: Given the localization of Mrpl44 to the matrix of the mitochondria and its association with the mitoribosome, we sought to determine if Mrpl44 is required for the expression of the mtDNA. We investigated the impact on mtDNA-encoded RNA and protein expression levels in NIH3T3 cells following perturbation of Mrpl44 levels. Knockdown of Mrpl44 by stably transduced shRNAs resulted in decreased levels of mtDNA-encoded RNA. 16S rRNA, ND2, ND4 and ND5 mRNA transcripts, in particular, were significantly decreased (Fig 3A). On the other hand, when Mrpl44 was overexpressed, mtDNA-encoded RNA was increased (S3 Fig). These results indicate that Mrpl44 affects the level of RNA within the mitochondria.


A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

Yeo JH, Skinner JP, Bird MJ, Formosa LE, Zhang JG, Kluck RM, Belz GT, Chong MM - PLoS ONE (2015)

Knockdown of Mrpl44 affects the expression of mtDNA-encoded genes.Protein and RNA were extracted from NIH3T3 knocked down (ORF) for Mrpl44 or expressing a control shRNA. (A) RNA expression of mitochondrial genes was analyzed by quantitative RT-PCR. Expression was normalized to β-actin. Statistical analysis was performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005). The mean +/- SEM of 3 independent experiments is shown. (B) Western blotting for the OXPHOS proteins COX IV and ND6, mitoribosome proteins Mrpl44, Mrpl11 and Mrps15. The mitochondrial protein Grp75 was analysed as a loading control. A representative of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519308&req=5

pone.0134326.g003: Knockdown of Mrpl44 affects the expression of mtDNA-encoded genes.Protein and RNA were extracted from NIH3T3 knocked down (ORF) for Mrpl44 or expressing a control shRNA. (A) RNA expression of mitochondrial genes was analyzed by quantitative RT-PCR. Expression was normalized to β-actin. Statistical analysis was performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005). The mean +/- SEM of 3 independent experiments is shown. (B) Western blotting for the OXPHOS proteins COX IV and ND6, mitoribosome proteins Mrpl44, Mrpl11 and Mrps15. The mitochondrial protein Grp75 was analysed as a loading control. A representative of three independent experiments is shown.
Mentions: Given the localization of Mrpl44 to the matrix of the mitochondria and its association with the mitoribosome, we sought to determine if Mrpl44 is required for the expression of the mtDNA. We investigated the impact on mtDNA-encoded RNA and protein expression levels in NIH3T3 cells following perturbation of Mrpl44 levels. Knockdown of Mrpl44 by stably transduced shRNAs resulted in decreased levels of mtDNA-encoded RNA. 16S rRNA, ND2, ND4 and ND5 mRNA transcripts, in particular, were significantly decreased (Fig 3A). On the other hand, when Mrpl44 was overexpressed, mtDNA-encoded RNA was increased (S3 Fig). These results indicate that Mrpl44 affects the level of RNA within the mitochondria.

Bottom Line: This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts.We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome.These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia; St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.

ABSTRACT
We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

No MeSH data available.