DOI: 10.5281/zenodo.17044290
title: Oxford Nanopore Direct RNA Sequencing datasets for detecting rRNA modifications in the Brassica oleracea mitoribosome

description: Oxford Nanopore Direct RNA Sequencing (DRS) was applied for the detection of rRNA modifications in the Brassica oleracea mitoribosome. A comparison between native rRNA transcripts and in vitro transcribed (IVT) rRNA transcripts that were devoid of any modification indicated systematic base-calling errors and/or variations in current intensities that led to the prediction of the modified nucleotides (Begik et al., 2018).

For Nanopore (DRS) library preparation, custom reverse transcription adapters (RTAs) containing Deeplexicon multiplexing barcodes (BC1, BC2 or BC3) were designed for sequence-specific ligation to the 3’-ends of B. oleracea mitochondrial rRNAs.

Basecalling and demultiplexing of ONT direct RNA sequencing data were performed by Guppy and Deeplexicon, respectively.

For the analysis and visualization of current intensities, a full description is available in the associated publication.

The Eventalign_18S.R Rscript was used to make the nanopore 18S signal analysis

The Eventalign_26S.R Rscript was used to make the nanopore 26S signal analysis

The raw data for the scripts are in the following folders:

- For 26S: 26S_Native.barcode01 and 26S_IVT.barcode03- For 18S: 18S_Native.barcode01 and 18S_IVT.barcode02

These folders contain the read alignment files output from minimap2 (in .bam format) and the eventalign files generated by the f5c software (in tsv format).

The FASTA folder contains the mitochondrial 18S and 26S rRNA gene reference sequence in fasta format
description: Publication information

Structural insights into maturation and translation of a plant mitoribosome

Vasileios Skaltsogiannis 2, Tan-Trung Nguyen 3, Nicolas Corre 2, David Pflieger 2, Todd Blevins 2, Yaser Hashem 3, Philippe Giegé 2*, Florent Waltz 1*

1. Biozentrum, University of Basel, Spitalstrasse 41, 4056 Basel, Switzerland.2. Institut de biologie de moléculaire des plantes CNRS, Université de Strasbourg, 12 rue du général Zimmer, 67084 Strasbourg, France3. Institut Européen de Chimie et Biologie, Université de Bordeaux, 2 rue R. Escarpit, F-33600 Pessac, France* Corresponding authors

 

Abstract

Ribosomes are key molecular machines that translate mRNA into proteins. Mitoribosomes are specific ribosomes found in mitochondria, which have been shown to be remarkably diverse across eukaryotic lineages. In plants, they possess unique features, including additional rRNA domains stabilized by numerous pentatricopeptide repeat proteins. However, the molecular mechanisms of translation by plant mitoribosomes remain largely unknown. Here, we use cryo-electron microscopy to provide a high-resolution structural characterization of the flowering plant mitoribosome, in translating and maturation states. The structure reveals the mitoribosome bound to tRNA in the peptidyl site, along with a segment of mRNA and a nascent polypeptide. Moreover, we identify an extensive set of ribosomal RNA modifications that we confirmed by nanopore sequencing. Additionally, we observe a late assembly intermediate of the small ribosomal subunit, in complex with the RsgA assembly factor. This reveals how a plant-specific extension of RsgA blocks the mRNA channel to prevent premature mRNA association before complete small subunit maturation. Our findings elucidate key aspects of translation in flowering plant mitochondria, revealing its distinct features compared to other eukaryotic lineages.