Project description:The goal of this analysis was to identify the ribosomal RNA content of the mitochondrial risome of Chlamydomonas reinhardtii green alga
Project description:Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells.
Project description:Ribosome profiling (RiboSeq) maps positions of translating ribosomes on the transcriptome. Here we optimized ribosome profiling for footprinting mitochondrial ribosomes, and profiled three human cell-lines - HEK293, a PDE12-/- knockout, and a deltaFLP 143B cybrid.
Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here, we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but employs duplex-specific nuclease as a convenient, bias-free, species-independent way to reduce rRNA contamination. Our protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream and overlapping open reading frames (ORFs). This feature also allows the identification of an alternative ribosome conformation evident during termination of protein synthesis. To test the effectiveness of DSN in ribosome profiling, we generated Ribo-Seq libraries from mouse tissue culture cells and from the green alga Chlamydomonas reinhardtii.
Project description:Mitochondria are essential components of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few, although essential, mitochondrial messenger RNAs still encoded by mitochondrial genomes. Here, we present the biochemical and structural characterization of the model green alga Chlamydomonas reinhardtii mitoribosome as well as the functional study of some of its Chlamydomonas specific components. We provide a single particle cryo-electron microscopy structure of Chlamydomonas mitoribosome and show how its core is composed by the assembly of 13 rRNA fragments encoded by separate non-contiguous gene pieces. While 5S rRNA was assumed to be absent from Chlamydomonas mitoribosome, a small rRNA representing a highly divergent 5S rRNA occurs in its central protuberance. In contrast, a gene piece that was believed to encode a fragment of rRNA encodes a small RNA that does not occur in the mitoribosome. Eleven novel proteins, mainly helical repeat proteins, including OPR, PPR and mTERF proteins occur in Chlamydomonas mitoribosome, revealing in particular the first structure of an OPR protein in complex with its RNA target. Reverse genetic studies reveal that the functions of some of the novel helical repeat proteins are required for mitoribosome biogenesis. The long studied p32 protein, whose function was related to several mitochondrial diseases in Human uniquely occurs as a constitutive ribosomal protein in the small subunit. Finally, cryo-electron tomography and biochemical studies show that Chlamydomonas mitoribosomes are exclusively attached to the mitochondrial inner membrane via two contact points mediated by Chlamydomonas specific novel proteins. One of them provides a direct interface between the exit of the peptide channel and the insertase Oxa1 for the insertion of nascent proteins in the mitochondrial inner membrane.
Project description:Ribosome profiling and RNAseq data on human BJ fibroblasts and cybrid cells using an adapted ribosome profiling protocol to improve detection of mitochondrial ribosome protected fragments 51-base length single read ribosome profiling data on human fibroblasts and cybrid cells using an adapted ribosome profiling protocol; and 51-base length single read RNAseq on polyA enriched RNA
Project description:In animals the organization of the compact mitochondrial genome and lack of introns have necessitated a unique mechanism for RNA processing. To date the regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. To understand the in vivo role of the endoribonuclease component of the RNase P complex, MRPP3, we created conditional knockout mice. Here we show that MRPP3 is essential for life, and heart and skeletal muscle-specific knockout leads to a cardiomyopathy early in life, indicating that it is the only RNase P enzyme in mitochondria. We show that RNA processing is required for the biogenesis of the respiratory chain and mitochondrial function. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-Seq enabled us to identify the in vivo cleavage sites of RNase P. Cleavage of the 5â² tRNA ends precedes 3â² end processing in vivo and is required for the correct biogenesis of the mitochondrial ribosomal subunits and mitoribosomal proteins that are differentially stabilized or degraded in the absence of mature rRNAs. Finally we identify that large mitoribosomal proteins can form a subcomplex on a precursor mt-RNA containing the 16S rRNA indicating that mitoribosomal biogenesis proceeds co-transcriptionally. Taken together our data show that RNA processing links transcription to translation via assembly of the mitoribosome. Total RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), TruSeq libraries produced in triplicate, sequenced and analysed for differential expression. Mitochondrial RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), PARE libraries produced in triplicate and sequenced for analysis of mitochondrial RNA processing.
Project description:Ribosome profiling and RNAseq data on human BJ fibroblasts and cybrid cells using an adapted ribosome profiling protocol to improve detection of mitochondrial ribosome protected fragments