Abstract
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.
Highlights
Telomeres are protective structures at the ends of linear chromosomes [1,2]
To date all telomerase RNA (TER) contain more than one telomeric repeat in their template, so that at least 2 of the same nucleotides are repeated at the beginning and the end of the template [25]
It was determined whether the sequences were conserved in the A. flavus genome, since A. oryzae and A. flavus are closely related [56,57]
Summary
The telomeric DNA consists of a tandemly repeated sequence, which varies in nucleotide composition and length depending on the organism. As cells replicate their DNA during each cell division cycle, their telomeres progressively shorten [3]. TERT proteins have been identified in genomes ranging from fungi to humans with conserved reverse-transcriptase and telomerasespecific protein motifs [6]. TERs have been identified from other ciliates [8,9,10,11,12,13], vertebrates [14], yeasts [15,16,17,18,19], plants [20], and very recently in filamentous fungi [21]. Since some filamentous fungi, such as the Aspergilli, possess extremely short and tightly regulated telomeres [22,23], they provide a unique model system to study telomere dynamics
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