Mapping of Mitochondrial RNA-Protein Interactions by Digital RNase Footprinting

Human mitochondrial DNA is transcribed as long polycistronic transcripts that encompass each strand of the genome and are processed subsequently into mature mRNAs, tRNAs, and rRNAs, necessitating widespread posttranscriptional regulation. Here, we establish methods for massively parallel sequencing...

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Bibliographic Details
Published in:Cell reports (Cambridge) 2013-11, Vol.5 (3), p.839-848
Main Authors: Liu, Ganqiang, Mercer, Timothy R., Shearwood, Anne-Marie J., Siira, Stefan J., Hibbs, Moira E., Mattick, John S., Rackham, Oliver, Filipovska, Aleksandra
Format: Article
Language:English
Subjects:
Gene Expression Regulation
Humans
Protein Footprinting - methods
Ribonucleases - genetics
Ribonucleases - metabolism
RNA - genetics
RNA - metabolism
RNA, Transfer - genetics
RNA, Transfer - metabolism
Transcription, Genetic
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Summary:Human mitochondrial DNA is transcribed as long polycistronic transcripts that encompass each strand of the genome and are processed subsequently into mature mRNAs, tRNAs, and rRNAs, necessitating widespread posttranscriptional regulation. Here, we establish methods for massively parallel sequencing and analyses of RNase-accessible regions of human mitochondrial RNA and thereby identify specific regions within mitochondrial transcripts that are bound by proteins. This approach provides a range of insights into the contribution of RNA-binding proteins to the regulation of mitochondrial gene expression. [Display omitted] •Mapping of mitochondrial RNA-protein interactions at a single-nucleotide level•Gene-specific knockdown identifies new and specific RNA binding sites of proteins•Identification of mitochondrial ribosomal footprints on RNAs•Detailed identification of mitochondrial RNA secondary structure in vivo Filipovska and colleagues describe methods for the analyses of RNase-accessible regions of the mitochondrial transcriptome using next-generation sequencing. They have combined these methods with gene-specific knockdowns to map mitochondrial RNA-protein interactions with single-nucleotide resolution and identify specific RNA binding sites. In addition, transcriptome-wide changes during stalled translation were identified and RNA structure was determined. The approaches provide a range of insights into the contribution of RNA-binding proteins to the regulation of gene expression, and they open the door for many additional discoveries.
ISSN:2211-1247
2211-1247
DOI:10.1016/j.celrep.2013.09.036