Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import

Abstract

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease.We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function

“…As illustrated in Figure 1A, the expression level of mitochondria-encoded protein ND5 and ND2 of mitochondrial complex I, COX I and COX II of mitochondrial complex IV were significantly decreased in MRPL10 knockdown cells, whereas nuclear-encoded mitochondrial proteins, such as NUDFS3, COX IV, and ATP5A had not changed. Consistent with previous findings which showed that mitochondrial translation was decreased globally upon MRPL12 depletion or Mrps34 mutation (Richman et al , 2015; Nouws et al , 2016). Next, mitochondrial complex activity was assayed as shown in Figure 1B.…”

“…In addition, we further defined MRPL10 is critical for mitochondrial function and mitochondrial fission by affecting mitochondrial protein synthesis and mitochondria dynamics. This is consistent with previous observation that knockdown of MRPL10 affect mitoribosome assembly (Nouws et al , 2016), and disrupting the mitoribosome intact by actinonin can block cell proliferation due to a mitoribosomal rescue pathway triggered by mitochondrial translation stress (Richter et al , 2013). The study also identifies the role of MRPL10 in the regulation of different signaling pathways, including mitochondrial complexes activity, cyclin B1/Cdk1 signaling, and mitochondria fission, which may help us to understand the mutual connection among these pathways.…”

“…In this study our results indicate that MRPL10 depletion decreases the phosphorylation of Drp1 at Ser616 by the inhibition of cyclin B1/Cdk1 activity, which lead to a significant mitochondrial clustering and fusion, suggesting the important roles of mitochondrial ribosomal proteins in mitochondrial homeostasis. Interestingly, MRPS29 (DAP3) knockdown can induce mitochondrial fragmentation, indicating that different mitochondrial ribosomal proteins may have multiple effects on the regulation of mitochondrial homeostasis (Nouws et al , 2016). The importance of mitochondrial fragmentation or excess mitochondrial fusion induced by the knockdown of distinct mitochondrial ribosomal proteins need to be tested, whether it is a rescue pathway for mitochondrial translation stress resistance or just a byproduct is still unknown.…”

“…For example, the Drosophila mRpL12 (Mitochondrial Ribosomal Protein L12) is required for CycD/Cdk4-induced cell growth (Frei et al , 2005). Human MRPL12 can bind and activate mitochondrial RNA polymerase (POLRMT), which may coordinate mitoribosome biogenesis and transcription in human mitochondria (Nouws et al , 2016). MRPS18-2 is involved in pRb control of the cell cycle through direct interaction with pR (Kashuba et al , 2008).…”

Estudo de proteínas que afetam a tradução mitocondrial em <i>Saccharomyces cerevisiae</i>.

“…na matriz e estão envolvidas na regulação da transcrição mitocondrial e na biogênese mitocondrial (NOUWS et al, 2016). …”