Abstract
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.
Highlights
Human mitochondrial DNA has limited coding capacity, having only 37 genes coding for 13 subunits of oxidative phosphorylation enzyme complexes, 22 transfer RNAs and 2 ribosomal RNAs
The results indicate that some mitochondrial RNA interacting proteins, in particular SUV3 and to a lesser extent GRSF1 and POLRMT, are more abundant in the XL sample without ethidium bromide (EtBr) compared to the XL sample with EtBr
Our analyses indicated that 4SU RNA labeling combined with mitochondrial isolation, subsequent crosslinking and poly(A) RNA isolation, scored best in terms of enrichment for mitochondrial RNA binding proteins
Summary
Human mitochondrial DNA (mtDNA) has limited coding capacity, having only 37 genes coding for 13 subunits of oxidative phosphorylation enzyme complexes, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs). In order to synthesize the 13 mtDNA encoded OXPHOS subunits, a unique mtDNA replication, transcription and translation system is in place Frontiers in Cell and Developmental Biology | www.frontiersin.org van Esveld et al. Mitochondrial Poly(A) RNA Interacting Proteome the combined action of mtDNA, its structural RNA components and as many as 250–300 nuclear-encoded gene products (Pearce et al, 2017) that are translated on cytosolic ribosomes and imported into mitochondria by dedicated outer- and inner-membrane machineries. Mitochondrial Poly(A) RNA Interacting Proteome the combined action of mtDNA, its structural RNA components and as many as 250–300 nuclear-encoded gene products (Pearce et al, 2017) that are translated on cytosolic ribosomes and imported into mitochondria by dedicated outer- and inner-membrane machineries The majority of these imported proteins are directly or indirectly involved in mitochondrial translation. A functional mitochondrial gene expression system requires the action of a large variety of mitochondrial RNA interacting proteins
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