ABSTRACT
In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome.
Subject(s)
Bacterial Toxins/genetics , Mitochondria/genetics , Mitochondrial Ribosomes/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Ribosomal, 16S/genetics , Ribonucleases/genetics , Bacterial Toxins/metabolism , Base Sequence , Biological Transport , Cell Engineering/methods , Cell Line , HEK293 Cells , Humans , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Mitochondrial Ribosomes/ultrastructure , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neurospora crassa/chemistry , Neurospora crassa/metabolism , Nucleic Acid Conformation , RNA Processing, Post-Transcriptional , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleases/metabolismABSTRACT
Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.
