Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation

Original Panther analysis tool data tables showing values and p -values for overrepresentation data sets. (XLSX 21 kb

Meiotic Insurance: Designing a system to study crossover control in yeast

Meiosis is a specialized form of cell division in which haploid gametes are produced from diploid progenitors. This reduction in ploidy results from proper meiotic chromosome segregation and is ensured by crossover recombination events. Given their importance, it is no surprise that crossover formation is regulated in most eukaryotes. Crossover assurance is a regulatory mechanism that helps to ensure that each pair of chromosomes gets at least one crossover during meiosis. We seek to better understand how crossover assurance works. To do so, we have developed a system in which crossover formation between a pair of chromosomes is restricted to a defined region. If crossover assurance functions in this context, then crossovers should frequently form in this defined region. Our experiments involve three yeast strains: Homolog: diploid Saccharomyces cerevisiae. Homeolog: Diploid S. cerevisiae, but with one copy of III derived from S. paradoxus and one from S. cerevisiae. Homo-meolog: The homeolog strain, but with the HIS4 region of the S. paradoxus III replaced with the corresponding S. cerevisiae sequence. S. cerevisiae and S. paradoxus are largely syntenic and have 80-90% sequence homology. This level of sequence divergence greatly reduces the incidence of meiotic crossing over. Thus, in the Homeolog strain chromosomes III will frequently fail to form crossovers. In the Homo-meolog strain, a defined region of homology surrounding HIS4 (a hotspot for meiotic recombination) exists in a chromosomal context of homeology. In the Homo-meolog strain, crossover assurance should result in a high incidence of crossover formation in the HIS4 region. By comparing the spectrum of meiotic recombination events in the HIS4 region in the three strains, we will gain insight into the means through which crossover assurance is enforced. These experiments are in the preliminary stage. Strain construction and data collection are ongoing, but our preliminary results demonstrate an elevated incidence of crossing over in the HIS4 region in the homo-meolog strain relative to both the homolog and homeolog strains. Spore viability patterns in the homo-meolog strain are not statistically distinguishable from that of the homolog strain, but are different from that of the homeolog strain. Taken together, these results suggest that the crossovers are targeted to the HIS4 region in the homo-meolog strain, possible through the action of a crossover assurance mechanism. Further analysis of the patterns of recombination in these strains may provide insight into the means through which this regulation is exerted

Applying thiouracil tagging to mouse transcriptome analysis

Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type-specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

Abstract Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction.

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells

Data from: Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction.

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells

HEK293T cell small RNA-seq

small RNA-seq reads from HEK293T cells mapped to human genome hg19