Identification of Arabidopsis mitochondrial ribosome proteome
ABSTRACT: In order to identify the comprehensive composition of Arabidopsis mitochondrial ribosome proteins, a strategy based on complementary approaches was used. Classical biochemical purification of ribosomes was combined with the immunoprecipitation of mitoribosomes using a specific Arabidopsis mitoribosome protein as a bait and quantitative proteomics. The immuno-purifications (IP) of the mitoribosome were performed on purified mitochondria from rPPR1-HA plant line (flowers as starting material). IPs were performed performed in different conditions (i.e. with 100, 400 or 600 mM KCl at the IP washing step or 800mM all along the IP). For each condition, experiments were performed in triplicates and IP proteins were identified by quantitative nano LC-ESI-MS/MS. Classic biochemical purification of mitochondrial monosomes (i.e. free ribosomes) was also performed. Starting from purified wild-type Arabidopsis mitochondria (cell culture as starting material), ribosomes were separated on high resolution 10-30% continuous sucrose gradients and mitoribosome containing fractions were analyzed by quantitative nano LC-ESI-MS/MS.
Project description:Polyribosomal fractions derived from P25 mice were the starting material for IPs of the FMRP protein and associated mRNA. Therefore we wanted to determine the relative abundance of all mRNAs in the starting pool for the IP as a denominator with which to compare the IPed material. [mRNA profiling]: We prepared polyribosomes on sucrose gradients in duplicate from 2 FVB WT mice, age P25, and purified RNA from polyribosomal fractions of each of the two gradients.
Project description:Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicate though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we used the ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosomes footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated to higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V where higher ribosome coverage corroborate with higher needs in specific subunits. In complex I respiratory mutants, a slight reorganization of mitochondrial mRNAs ribosome association was detected involving an increase in ribosome densities on certain ribosomal protein encoding transcripts and a reduction in the translation of a few complex V mRNAs. Altogether, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding of translation in higher plant mitochondria. Overall design: In this study, 2 Arabidopsis mutants are compared to the wild type using duplicates for the Ribosome Profiling experiment. RNASEQ: was conducted to observe the editing rates of Col0 transcripts. RIBOSEQ: was conducted to observe the ribosome occupancy on each transcript of wild type and mutants.
Project description:We evaluated the performance of 5 library prep protocols by using total mRNA and IP RNA of mouse liver,we found all the 5 library preparation kits detect more enrichment effects than depletion effect. The profiles being generated by SMARTer kit is different than all other kits. Overall design: Cell-type specific RNA from livers of P30 mice was obtained using the RiboTag protocol. Briefly, liver tissue expressing HA-tagged ribosomes in specific cells was homogenized followed by immunoprecipitation of the tagged ribosomes using an HA antibody. The RNA was extracted from the immunoprecipitated ribosomes to obtain cell-type specific enriched transcripts (IP samples). An aliquot of the homogenized tissue before immunoprecipitation was used for total RNA extraction (input samples).
Project description:We report that phosphorylated ribosomes can be immunoprecipitated from mouse brain homogenates, resulting in enrichment of transcripts expressed in activated neurons. Mice were either injected with a concentrated salt solution or vehicle, hypothalami dissected, and phosphorylated ribosomes immunoprecipitated. RNA was sequenced from the input and IP for each condition (4 samples total).
Project description:Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with different selectivity for translating subpools of transcripts including those controlling metabolism, the cell cycle, and development. As a paradigm example, we show that mRNAs enriched in binding to RPL10A/uL1-containing ribosomes require RPL10A/uL1 for their efficient translation. Within several of these transcripts, we find this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression. Overall design: To identify the transcripts specifically translated by ribosomes demarcated by RPL10A or RPS25, we enriched for RPL10A or RPS25-containing ribosomes by immunoprecipitation (IP), and performed RPL10A-Ribo-Seq and RPS25-Ribo-Seq (2 replicates each), with the control being total Ribo-Seq (2 replicates each). To further assess the translation efficiencies of mRNAs upon Rpl10a knockdown at genome-wide scale, we performed RNA-Seq (2 replicates each) of purified RNAs from combined medium and heavy polysome fractions containing the most actively translating ribosomes (>=4 ribosomes along a mRNA molecule), and from all other fractions containing the free ribonucleoproteins (RNPs), 40S/60S ribosomal free subunits, 80S/monosomes as well as light polysomes (2-3 ribosomes per mRNA molecule).
Project description:Increasing evidence indicates that ribosomes are heterogeneous and perhaps dynamic, in contrast to the classical view of them as constitutive machinery for protein synthesis [1-11]. In Drosophila melanogaster, nine ribosomal protein genes are each present in two paralogs, and in many of these cases one of the paralogs is primarily expressed in germline tissues [12-14]. Among these are two genes encoding ribosomal protein S5, called RpS5a and RpS5b. We demonstrate that RpS5b is specifically required for oogenesis. Females lacking RpS5b produce ovaries with polarity defects, fused egg chambers, and over-proliferation of posterior follicle cells. Oogenesis ceases at stage 8-9, and widespread apoptosis follows. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting RpS5a in an RpS5b mutant background worsens the RpS5b phenotype and causes complete germline lethality. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, proteome comparison of wildtype and RpS5b ovaries revealed that RpS5b mitochondria were depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration. As observed by immunohistochemical staining and transmission electron microscopy, RpS5b nurse cell mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We also observed that RpS5b ovaries have elevated ROS, consistent with mitochondrial dysfunction. We conclude that RpS5b-containing ribosomes preferentially associate with mRNAs encoding mitochondrial components and serve an essential function in oogenesis. Overall design: The mRNAs associated to either RpS5b and RpS5a were identified by co-IP followed by deep sequencing. The mRNAs enriched to either of the two parologs of RpS5, when applicable, were analyzed in three genetic backgrounds, wildtype, RpS5b-null and the ovary with germline RpS5a knocked down by RNAi.
Project description:Affymetrix U133 Plus 2.0 arrays were hybridized with varying amounts of starting material to determine whether different measurements of gene expression were observed. Keywords: Affymetrix starting material study Overall design: 1 ug, 2, ug, 5, and 10 ug of Stratagene universal human reference RNA was used to setup a series of reactions which were subsequently hybridized to Affymetrix U133 Plus 2.0 arrays. 1 ug samples were performed in triplicate and the entire experiement was repeated.
Project description:Polysome-profiling is commonly used to study translatomes, i.e. transcriptome-wide patterns of translational efficiency. The standard approach for collecting efficiently translated polysome-associated RNA results in laborious extraction of RNA from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts such as primary cells or frozen tissues. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for mRNAs associated with >3 ribosomes in only one or two fractions, thereby reducing sample handling 5-10 fold. The technique can be applied to frozen tissue samples from biobanks, and generates polysome-associated RNA with a quality reflecting the starting material. When coupled with smart-seq2, a single-cell RNA sequencing technique, translatomes from small tissue samples can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associated with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation to maintain protein levels constant. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in bio banks. Overall design: We used two cell lines that differ in their p53 status (HCT-116 p53+/+ and HCT-116 p53-/-) and serum-starved them (16 h). We prepared cytosolic lysates from 6 plates (15 cm) from each cell type and divided the lysates equally between the optimized non-linear gradient and the linear gradient groups. We then collected fractions corresponding to mRNA associated with >3 ribosomes from the linear gradient, peak 0 and +1 from the optimized non-linear gradient as well as the cytosolic input. We repeated the experiment four times.
Project description:Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1. Overall design: 6xFLAG-3xHIS-AGO1 (6F3H-AGO1) and 6xFLAG-3xHIS-AGO2 (6F3H-AGO2) proteins were immunoprecipitated from C. higginsianum mycelia tissue, along with a non-tagged protein control. RNAs isolated in the IP fraction were used as the starting material for the small RNA library protocol. The input and IP fractions of three independent transformants containing 6F3H-AGO1, two independent transformants containing 6F3H-AGO2, and one replicate of wild-type (mock-IP) were sequenced.