Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5′ splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

Telomeres, the protective caps of linear chromosomes, have been shown to vary in length between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. In the filamentous fungus Aspergillus nidulans, which has several different cell types, the telomere lengths in vegetative hyphae and spores are invariant. No study has determined the telomere length of the sexual ascospores. We report the development of a PCR technique to measure the telomere length in A. nidulans, including the meiotic cells. This novel technique that we call “telomere‐anchored PCR,” measures telomere length using a fraction of the DNA required for terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. Furthermore, an increase in telomere length is not necessary for meiosis in this organism. When the hyphal telomeres were measured in a telomerase reverse transcriptase knockout strain, decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types, is sensitive enough to measure them when exceptionally short, and might be utilized in other organisms.