Project description:To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we first benchmarked for in vivo use using cortical pyramidal neurons, an abundant population.
Project description:To illuminate the molecular mechanisms driving neuronal differentiation we generated a mouse line amenable to mapping miRNA-target interactions in rare cell types. Biochemical approaches to purify AGO2-miRNA-target complexes have successfully mapped MTIs in abundant populations of neurons. However, due to their technical complexity and high background, these approaches are not suitable for mapping interactions in rare cell populations such the many neuronal subtypes that compose the mammalian brain. We therefore generated a mouse line with a conditional SpyTag3, which is small and offers near-infinite affinity for pull-downs, in the endogenous Ago2 gene. We then developed a method Spy3-AGO2 pull-down and sequencing (SAPseq), which we have used to accurately map miRNA-target interactions in developing Purkinje cells, a rare population of cells in the cerebellum.
Project description:Studies have demonstrated that T6B can selectively de-repress miRNA targets in non-neuronal cells. However, since neurons may have a different expression profile of miRNA pathway genes as well as other binding partners of TNRC6, we sought to validate the intended function of in neurons. The first step was to identify bona fide miRNA targets in PNs, we performed AGO2 crosslinking and immunoprecipitation followed by sequencing (CLIPseq).
Project description:Here, seeking to gain insight into the array of transcripts engaged with miRNAs in human brain, we performed HITS-CLIP to profile transcriptome-wide Ago2:RNA interactions in a panel of eleven post-mortem adult human brain samples harvested from adult motor cortex and cingulate gyrus, regions associated with movement and psychiatric disorders . High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) Eleven post-mortem adult human brain tissues were subjected to ultraviolet radiation to crosslink proteins with nucleic acids, and HITS-CLIP was performed as previously described by Chi et al Nature. 2009 Jul 23;460(7254):479-86 using an anti-Ago2 polyclonal antibody and some additional modifications.
Project description:Here, seeking to gain insight into the array of transcripts engaged with miRNAs in human brain, we performed HITS-CLIP to profile transcriptome-wide Ago2:RNA interactions in a panel of eleven post-mortem adult human brain samples harvested from adult motor cortex and cingulate gyrus, regions associated with movement and psychiatric disorders . High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) Eleven post-mortem adult human brain tissues were subjected to ultraviolet radiation to crosslink proteins with nucleic acids, and HITS-CLIP was performed as previously described by Chi et al Nature. 2009 Jul 23;460(7254):479-86 using an anti-Ago2 polyclonal antibody and some additional modifications.
Project description:Mapping of microRNA (miRNA) targets using AGO2 CLIPseq and HEAPseq has provided major insight into the function of miRNAs in various systems. HEAPseq is a powerful method because it allows for cell type-specific mapping of miRNA targets and relies on a mouse line harboring a conditional HaloTag in the endogeous Ago2 locus. However, the homozygous mice are not viable, suggesting that the insertion of the large (>1 kb) HaloTag conditional cassette in the Ago2 gene promoter region, or appending the HaloTag domain to the AGO2 protein, is deleterious to some aspects of AGO2 function. To overcome this limitation, we created tagged AGO2 mouse and mESC lines designed for minimal perturbation of miRISC function. We used a cleavable SpyTag3, which rapidly forms a covalent bond with its ligand SpyCatcher3 and is ten times smaller than HaloTag (3 vs 33 kDa)36. To minimize any perturbation on AGO2 expression and protein structure, constitutive Spy3-AGO2 mice and mESC lines were created by inserting the SpyTag3 coding sequence into exon 2 of the Ago2 gene, which is separated from the promoter region by a 38 kb intron and encodes a small unstructured region of AGO2 – the only part of the protein sequence that is not perfectly conserved between mice and humans. We then developed a method SAPseq, based on HEAPseq and benchmarked it first using mESCs.
Project description:We deciphered Ago2:RNA interactions in post-mortem human heart tissues using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts.
Project description:We deciphered Ago2:RNA interactions in post-mortem human heart tissues using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Profiling AGO2-associated RNAs in cardiac tissues obtained from 6 donors with heart diseases of differing etiologies