Aging disrupts spatiotemporal regulation of germline stem cells and niche integrity.

A major factor driving stem cell decline is stem cell niche aging, but its molecular mechanism remains elusive. We use the Caenorhabditis elegans distal tip cell (DTC), the mesenchymal niche that employs Notch signaling to regulate germline stem cells (GSCs), as an in vivo niche aging model and delineate the molecular details of the DTC/niche aging process. Here, we demonstrate that a drastic decrease in C. elegans germline fecundity, which begins even in early adulthood, is mainly due to an age-induced disruption in spatial regulation of Notch-dependent transcription in the germline combined with a moderate reduction in Notch transcription at both tissue and cellular levels. Consequently, the Notch-responsive GSC pool shifts from the distal end of the gonad to a more proximal region, disrupting the distal-to-proximal germline polarity. We find that this GSC pool shift is due to a dislocation of the DTC/niche nucleus, which is associated with age-induced changes in the structure and morphology of the DTC/niche. Our findings reveal a critical link between physiological changes in the aging niche, their consequences in stem cell regulation, and germline tissue functions.

A Split NanoLuc Reporter Quantitatively Measures Circular RNA IRES Translation.

Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5' untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA.