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:These are the ribosomal subunit fractions from the polysome gradients. investigating effect of heat shock on procyclic-form trypanosomes.
Project description:affy_tour_2012-02 - Identification of transcripts that are addressed to traduction in imbibed seeds in relation with dormancy: comparison of the translatome in Dormant versus Non-Dormant seeds -- At harvest seeds are dormant. They stay dormant if they are stored at -20°C (D) and become non-dormant (ND) if they are stored 2 months at +20°C. Polysomal fractions were purified on sucrose gradients from sunflower axis isolated from dormant and non-dormant seeds imbibed at 10°C during 3h, 15h or 24h. - These fractions allow to identify the transcripts addressed to translation (translatome) during the seed imbibition process (3, 15 and 24h) - The translatome of 2 types of seeds are compared: Dormant vs Non-Dormant at the 3 time points. 18 arrays - SUNFLOWER; time course,treated vs untreated comparison
Project description:The Fragile X Mental Retardation Protein, FMRP, is thought to regulate the translation of a specific set of neuronal mRNAs on polyribosomes. Therefore, we prepared polyribosomes on sucrose gradients and purified mRNA specifically from these fractions, as well as the total mRNA levels, to determine whether a set of mRNAs might be changed in its % association with polyribosomes in the absence of FMRP in the KO mouse model. No significant differences were found, other than the Fmr1 transcript itself, in total mRNA levels or % polyribosome association that withstood multiple test correction, in P7 Fmr1 KO mouse cerebral cortex compared with WT littermates.. We prepared polyribosomes on sucrose gradients from 6 littermate pairs of Fmr1 KO and WT littermates (FVB background, P7 males, cerebral cortex) and purified RNA from both polyribosomal fractions and input to the gradient, reflecting total mRNA levels for comparison.
Project description:Genome-wide translational profiling of rng3-65 compared to wild type cells. We used sucrose gradients to separate RNAs according to the number of associated ribosomes (a surrogate for translational efficiency). Preparation of the extracts and fractionation was carried out as described in Lackner et al, 2007 (Mol Cell 26(1):145-55). The fractions were pooled into four groups (1 closest to the top, i.e. not associated with ribosomes and 4 closest to the bottom, i.e., associated with polysomes). RNA was extracted from the pools and the corresponding pools from wild type and mutant cells were directly compared using DNA microarrays. Changes in translation are expected to alter the number of ribosomes associated with specific transcripts, and therefore result in a redistribution of the RNAs across the different fractions.
Project description:Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2-18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.
Project description:The purpose of this study was to compare changes in translation (using Gradient Encoding, described below) to changes in mRNA abundance. Lysates of wildtype v-Abl transformed pre-B cells harvested before and after 12 hours of treatment either with 2.5 uM imatinib, a v-Abl kinase inhibitor, or 10ng/mL (10.9 nM) rapamycin, an mTOR inhibitor, were fractionated by sedimentation through linear sucrose gradients. Gradient fractions were encoded such that the mRNA from successive fractions was labeled with increasing ratios of Cy5 to Cy3. mRNAs derived from fractions in the lighter portion of the gradient therefore have a lower Cy5 to Cy3 ratio, whereas those deeper in the gradient have a higher Cy5 to Cy3 ratio. The ratio of Cy5 to Cy3 for each mRNA therefore reflects its average position within the gradient. We thus encoded the sedimentation rate of each mRNA across the entire gradient. The resulting ratios were quantitatively measured for each mRNA species by hybridization to DNA microarrays, and related to the 260 nm absorbance peaks representing different numbers of ribosomes bound per mRNA Compound Based Treatment: wildtype v-Abl transformed pre-B cells were treated with imatinib mesylate (IMA), rapamycin (RAP) or nothing (NONE)