A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome.

In order to synthesize the 13 oxidative phosphorylation proteins encoded by mammalian mtDNA, a large assortment of nuclear encoded proteins is required. These include mitoribosomal proteins and various RNA processing, modification and degradation enzymes. RNA crosslinking has been successfully applied to identify whole-cell poly(A) RNA-binding proteomes, but this method has not been adapted to identify mitochondrial poly(A) RNA-binding proteomes. Here we developed and compared two related methods that specifically enrich for mitochondrial poly(A) RNA-binding proteins and analyzed bound proteins using mass spectrometry. To obtain a catalog of the mitochondrial poly(A) RNA interacting proteome, we used Bayesian data integration to combine these two mitochondrial-enriched datasets as well as published whole-cell datasets of RNA-binding proteins with various online resources, such as mitochondrial localization from MitoCarta 2.0 and co-expression analyses. Our integrated analyses ranked the complete human proteome for the likelihood of mtRNA interaction. We show that at a specific, inclusive cut-off of the corrected false discovery rate (cFDR) of 69%, we improve the number of predicted proteins from 185 to 211 with our mass spectrometry data as input for the prediction instead of the published whole-cell datasets. The chosen cut-off determines the cFDR: the less proteins included, the lower the cFDR will be. For the top 100 proteins, inclusion of our data instead of the published whole-cell datasets improve the cFDR from 54% to 31%. We show that the mass spectrometry method most specific for mitochondrial RNA-binding proteins involves ex vivo 4-thiouridine labeling followed by mitochondrial isolation with subsequent in organello UV-crosslinking.

Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure.

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

The hexameric structure of the human mitochondrial replicative helicase Twinkle.

The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity.