Distinct Technique Reveals Telomerase Rna is Not Exported from the Nucleus in Aspergillus Nidulans

Abstract

Telomeres are protein-nucleotide sequences at the ends of chromosomes that prevent the loss of genetic information during cell replication. The telomerase holoenzyme includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Biogenesis of telomerase occurs in the cytoplasm in Saccharomyces cerevisiae and the nucleus in mammalian cells. The structure of TER in the filamentous fungus Aspergillus nidulans is partially conserved in S. cerevisiae and partly in mammalian cells, but whether TER migrates to the cytoplasm for telomerase biogenesis is not known in A. nidulans. We used this fungi’s ability to form a multi-nucleate state and the heterokaryon rescue technique to determine the functional localization of TER, and subsequently infer the location of telomerase biogenesis. We assess the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA product for TER exits the nucleus. To perform the heterokaryon test, we generated a TER knockout A. nidulans strain ( ΔTER ) and analyzed whether heterokaryons formed, as heterokaryon formation would rely on TER export from the nucleus. This process was repeated for TERT as a control ( ΔTERT ). While the ΔTERT control formed heterokaryons, we found that the ΔTER strain consistently failed to form heterokaryons but instead formed diploids, which was verified by PCR and DAPI stain analysis of nuclear size and stain intensity. The lack of heterokaryon formation in ΔTER condition cells strongly suggests TER remains within nuclei and that telomerase assembly occurs within the nucleus, a scenario that is similar to mammals but not yeast. These results further suggest the potential of A. nidulans as a useful model organism to study telomerase assembly and activity mechanisms.

Keywords: Telomerase RNA, Telomere, Aspergillus nidulans, Heterokaryon Rescue Technique, Diploids, Nuclear Export