Dataset Information

Unraveling the complexities of mitochondrial supercomplexes by mass spectrometry-based proteomics

ABSTRACT: It is now accepted that mitochondrial respiratory complex subunits assemble in supercomplexes. However, studying supercomplexes has typically relied upon antibody-based protein quantification, often limited to a single subunit per respiratory complex. To provide a deeper insight into mitochondrial plasticity and supercomplex formation, we combined Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and high-resolution mass spectrometry-based proteomics in sedentary and exercise-trained skeletal muscle. We quantified 422 mitochondrial proteins within ten bands. Our analyses revealed the often-unaccounted presence of complex II and V subunits within the majority of supercomplexes. Upon exercise-induced mitochondrial biogenesis, non-stoichiometric changes in subunits and their incorporation into supercomplexes were apparent. Finally, we uncovered the dynamics of supercomplex-related assembly proteins and mtDNA-encoded subunits within supercomplexes, as well as the high-molecular mass complexes of ubiquinone biosynthesis enzymes and Lactb, a mitochondrial-localized obesogenic polymer. Thus, combining BN-PAGE and mass spectrometry to comprehensively analyze respiratory supercomplexes illuminates previously undetectable complexity in mitochondrial plasticity.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle, Muscle Cell Differentiation

SUBMITTER: atul shahaji deshmukh

LAB HEAD: Atul S Deshmukh

PROVIDER: PXD016289 | Pride | 2021-05-17

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20170324_QE6_nLC4_ASD_SA_C2C12_L1.raw	Raw
	20170324_QE6_nLC4_ASD_SA_C2C12_T1.raw	Raw
	20170324_QE6_nLC4_ASD_SA_WTR_L1.raw	Raw
	20170324_QE6_nLC4_ASD_SA_WTR_L2.raw	Raw
	20170324_QE6_nLC4_ASD_SA_WTR_L3.raw	Raw

Items per page:

1 - 5 of 101

Similar Datasets

Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5â² triphosphate characteristic of transcription start site and possess U-tail indicative of 3â² processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3â²-5â² DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3â² processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3â²-5â² degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5â² extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation. 1. we first sequenced guide RNA precursor (Gel-fractioned total cellular RNA 600-1500nt was used) transcripts from three replicates to study the their tail features and also validate observation of sense/antisense accumulation upon perturbation of pre-processing complex based on few cases. 2. we then sequenced mitochondrial small RNA and built a reference for small RNAs using our custom algorithm. We then took the mitochondrial small RNA data and uncovered the sense/antisense pair. 3. We then usedÂ CLIP-Seq data toÂ investigateÂ in vivo binding sitesÂ and, together with RNA IP-Seq data to understand what determine the relative abundance of sense and antisense pair of the duplex. 4. We used sequenced small mitochondrial RNA in different RNAi experiments (For RNAi experiments, 30-70nt RNA fraction was gel-isolated from total cellular RNAs) to understand which protein will affect the Utail length in mature small mitochondrial RNA.

Dataset Information

Unraveling the complexities of mitochondrial supercomplexes by mass spectrometry-based proteomics

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets