Characterization of Telomeric Repeat-Containing RNA (TERRA) localization and protein interactions in Primordial Germ Cells of the mouse
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.\n\nResearch reported in this publication was supported in part by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number K99HD090289 to M.A.B-E. A seed grant from the Cornell Center for Reproductive Genomics to M.A.B-E, using funds obtained as part of the NICHD National Centers for Translational Research in Reproduction and Infertility (NCTRI), award number P50HD076210 to P.E.C. and Empire State Stem Cell Fund through New York State Department of Health Contract # C30293GG. Imaging data was acquired through the Cornell University Biotechnology Resource Center, with NSF funding #1428922 for the shared Zeiss Elyra Microscope. NIH SIG 1S10 OD017992-01 grant support the Orbitrap Fusion mass spectrometer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.