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A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

ABSTRACT: A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs) relative to spontaneously differentiated cells (SDCs). Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide) upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

SUBMITTER: Gopalakrishna-Pillai S

PROVIDER: S-EPMC3115941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse ?-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (G??-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.

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A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

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