Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids

Abstract

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.

Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies

“…It is tempting to speculate that Dss1 supports a similar directed turnover of damaged mRNAs. Interestingly, we observed that Suv3 is a component of the nucleoid-MIOREX assembly and not part of the peripheral complexes, in line with previous data showing a preferential nucleoid interaction (Bogenhagen et al, 2014). Because Suv3 and Dss1 have to interact for efficient degradation (Malecki et al, 2007), this observation suggests a functional cycle of translation: MIOREX complexes adjacent to the nucleoid are charged with newly transcribed pre-mRNAs.…”

“…These RNA granules also contain RNA-binding proteins, methyltransferases, and ribosomal proteins, therefore likely representing the nucleoid-MIOREX complexes identified here. In addition to a role of the RNA granules in organizing the early steps of gene expression, it was demonstrated that also ribosome assembly occurs in proximity to the nucleoid (Bogenhagen et al, 2014;He et al, 2012). Because of technical difficulties in working with mitochondrial ribosomes, it was not known how the fully assembled ribosome is related to these structures.…”

Assignment of 2′-O-Methyltransferases to Modification Sites on the Mammalian Mitochondrial Large Subunit 16 S Ribosomal RNA (rRNA)

“…Human NOA1 (hNOA1), also known as MTG3 or C4orf14, is associated with the nucleoid and is necessary for SSU assembly (61)(62)(63). We have also identified ERAL1 in nucleoids (29,64). ERAL1 is a GTPase that functions as an RNA chaperone, binding to the TFB1M-mediated dimethylation region of 12 S rRNA (65,66), potentially folding the RNA into the proper conformation needed for SSU assembly.…”

“…In addition to finding methyltransferases localized near nucleoids, we have recently shown that newly synthesized MRPs are localized in the nucleoid complex to encounter newly synthesized rRNA using pulse-chase stable isotope labeling by/with amino acids in cell culture labeling (29). It is also known that GTPases are important for mitoribosome assembly, just as they are for bacteria (60).…”

“…Our understanding of the mitoribosome structure is improving with advancements in cryo-EM technology (12, 24 -28), but the assembly process has received little attention. We have recently used microscopy and stable isotope labeling by/with amino acids in cell culture (SILAC) proteomic analysis to show that a subset of newly synthesized mitochondrial ribosomal proteins participates in the early stages of ribosome assembly at the nucleoid (29).…”