Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and telomeric repeat-containing RNA (TERRA). Proposed TERRA functions are wide ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the presence and role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization, we identify TERRA transcripts in female PGCs at 11.5, 12.5, and 13.5 days postcoitum. In male PGCs, the earliest detection TERRA was at 12.5 dpc where we observed cells with either zero or one TERRA focus. Using qRT-PCR, we evaluated chromosome-specific TERRA expression. Female PGCs showed TERRA expression at 11.5 dpc from eight different chromosome subtelomeric regions (chromosomes 1, 2, 7, 9, 11, 13, 17, and 18) while in male PGCs, TERRA expression was confined to the chromosome 17. Most TERRA transcription in 13.5 dpc male PGCs arose from chromosomes 2 and 6. TERRA interacting proteins were evaluated using identification of direct RNA interacting proteins (iDRiP), which identified 48 in female and 26 in male protein interactors from PGCs at 13.5 dpc. We validated two different proteins: the splicing factor, proline-and glutamine-rich (SFPQ) in PGCs and non-POU domain-containing octamer-binding protein (NONO)
Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and Telomeric Repeat-Containing RNA (TERRA). Proposed TERRA functions are wide-ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization (RNA-FISH) we identify TERRA in PGCs soon after these cells have migrated to, and become established in, the developing gonad. RNA-FISH showed the presence of TERRA transcripts in female PGCs at 11.5, 12.5 and 13.5 days post-coitum. In male PGCs, however, TERRA transcripts are observable from 12.5 dpc. Using qPCR we evaluated chromosome-specific TERRA expression, and demonstrated that TERRA levels vary with sex and gestational age, and that transcription of TERRA from specific chromosomes is sexually dimorphic. TERRA interacting proteins were evaluated using Identification of Direct RNA Interacting Proteins (iDRiP) which identified 48 in female and 26 in male protein interactors specifically within nuclear extracts from PGCs at 13.5 dpc. We validated two different proteins the splicing factor, proline- and glutamine-rich (SFPQ) in PGCs and Non-POU domain-containing octamer-binding protein (NONO) in somatic cells. Our results show that, TERRA interacting proteins are determined by sex in both PGCs and somatic cells. Taken together, our data indicate that TERRA expression and interactome during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.
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