Project description:rs10-01_rrm - quadruple rrm experience - What is the role of the RRM protein family in plants? Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation. Keywords: normal vs transgenic comparison 2 dye-swap - CATMA arrays
Project description:rs10-01_rrm - quadruple rrm experience - What is the role of the RRM protein family in plants? Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation. Keywords: normal vs transgenic comparison
Project description:rs09-03_zf1 - zf1 experiment - Transcriptome analysis of mutants for novel AGO-hook type proteins in Arabidopsis thaliana - Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation Keywords: normal vs transgenic comparison 4 dye-swap - CATMA arrays
Project description:rs09-03_zf1 - zf1 experiment - Transcriptome analysis of mutants for novel AGO-hook type proteins in Arabidopsis thaliana - Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation Keywords: normal vs transgenic comparison
Project description:During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs or target mRNAs. We refer to our method as Ago protein Affinity Purification by Peptides (Ago-APP). Comparison of small RNA lengths in total RNA and APP-enriched RNA samples
Project description:During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21Cip1 and Drosophila Dacapo proteins. Both the CycE and Cip/Kip proteins are under elaborate control via protein degradation, mediated by the Cullin-Ring-Ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCFFbxw7/Ago targets phosphorylated CycE while p21Cip1 and Dap are targeted by the CRL4Cdt2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago (Fbxw7) mediated CycE degradation and of Dap/p21Cip1 degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. In contrast, expression of PIP degron mutant Dap/p21Cip1 transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.
Project description:During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs or target mRNAs. We refer to our method as Ago protein Affinity Purification by Peptides (Ago-APP).
Project description:Methionine sulfoxide reductase (Msr) is an important antioxidant enzyme. The mammalian Msr family has four members: MsrA, MsrB1, MsrB2, and MsrB3. We generated a mouse strain with deletion of all four Msr proteins. Deletion was confirmed by Western blotting. The quadruple knockout mouse is viable, and growth, development, and fertility appear normal. However, they are unexpectedly resistanct to oxidative stresses. We therefore determined the RNA expression profile in liver and heart of wild-type and quadruple knockout mice.
Project description:Small RNA-dependent pairing of AGO/PIWI-containing effector complexes to chromatin-bound nascent transcripts has been proposed as a universal mechanism for guiding RNA-mediated TGS in eukaryotes. Likewise, Pol V-dependent transcripts have been implicated in the targeting of AGO4 to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we show that the AGO hook platforms of PolV and SPT5L, another component of the transcriptional complex, are functionally redundant yet essential for RdDM at a genome-wide level. Synthesis of Pol V transcripts is uncoupled from AGO4 recruitment in AGO hook-minus plants, challenging the prevailing RNA-based mechanism of AGO4 targeting to chromatin in RdDM. Transcription is essential to lock the PolV transcription complex into a stable and productive DNA-bound state potentiating interactions of AGO4 with DNA. Consistent with this idea, laser UV-assisted crosslinking shows specific AGO4-DNA interaction at RdDM loci, suggesting a revised model for Pol V-mediated DNA methylation in plants, which explains the exquisite specificity of methylation.
