Identification of Telomerase RNAs from Filamentous Fungi Reveals Conservation with Vertebrates and Yeasts

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 59 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

Important pseudoknots are conserved in the filamentous fungi.

<p><i>A. oryzae/A. flavus</i> and <i>A. nidulans</i> are shown in comparison to yeast and human sequences<b>.</b> Red nucleotides indicate U-A·U base triples in addition to nucleotides fitting the conserved proposed pseudoknot consensus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>. Numbered nucleotides for the <i>A. oryzae/A. flavus</i> pseudoknot are specific to <i>A. oryzae</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661.s003" target="_blank">Figure S3</a> for additional Aspergilli pseudoknots.</p

Phylogenetic-based secondary structure prediction revealed organization and functional elements conserved with other TER sequences.

<p>Boxed regions indicate similarity of the <i>A. oryzae</i> TER to other TERs: blue-yeast; red-vertebrates, green-similarity to both yeast and vertebrate. Red nucleotides indicate conservation across all 13 of the Aspergillus TERs.</p

The 3′ end of the Aspergilli TERs contain conserved elements that function in TER processing.

<p>The Sm site is slightly variable, while the 5′ splice site and branch point are much more conserved within the Aspergilli. Numbers within the sequence indicate the linker nucleotides between the 5′ splice site and the branch point. Asterisks indicate conserved nucleotides in all 13 species.</p

A single transcribed sequence with a putative telomerase RNA template is identified in <i>A. oryzae</i>.

<p>A. RNA was reverse transcribed using one forward primer to amplify from the template towards the 5′ end (green). Different reverse primers were used to determine roughly where the transcript ended (green indicates RT-PCR product was present, red indicates RT-PCR product does not extend that far). Likewise, a reverse primer was used to amplify from the template to the 3′ end (purple). B. Sets of three products were analyzed by gel electrophoresis: RT-PCR reactions without RNase (−), reactions with RNase (+), and PCR reactions where genomic DNA was used instead of RNA (D). The set of three reactions is labeled by the genomic sequence (e.g. “H”), the strand orientation of the first primer (e.g. “3″) and the designation of the opposite primer of the pair (e.g. “J”). Alpha-tubulin primers on either side of an intron were used as a control, where an excised intron results in smaller products in the (−) lane than in the (D) lane. C. RLM-RACE results for the 3′ end (lanes 1 and 2) and the 5′ end (lanes 5 and 6). D. The sequence in green is the anticipated template. The sequences in blue were the primers used for the RT-PCR reaction that yielded products. Asterisks indicate ends that were determined by sequence analysis, some of which were redundant at the 3′ end.</p

Conservation of the three-way junction.

<p><i>A. oryzae</i>/<i>A. flavus</i>/<i>A. soaje</i> and <i>A. nidulans</i> are shown in comparison to yeast and human sequences. For the yeast and human sequences the same coloring scheme is used from Gunisova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>. For the Aspergilli TERs nucleotides in blue are conserved for 12 of 13 Aspergilli. Combining information from the TERs examined by Gunisova et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Gunisova1" target="_blank">[16]</a>, the nucleotides in red are conserved in 63 of 68 TERs examined. Numbered nucleotides for the <i>A. oryzae/A. flavus/A. sojae</i> three-way junction are specific to <i>A. oryzae</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661.s004" target="_blank">Figure S4</a> for additional Aspergilli three-way junctions.</p

Templates and template boundaries are conserved in the Aspergilli TERs.

<p>The TER template must contain a short repeated sequence at its 5′ and 3′ ends for alignment with the telomere end. This information allowed us to identify or confirm what telomeric repeats the template sequences of these organisms could synthesize, depending on whether the telomeric repeat sequence was already identified. For example, in <i>A. carbonarius</i> the template sequence begins with UAA at the 3′ end and ends with UAA at the 5′ end of the template, which would synthesize TTAGGG. It is unknown whether A. fumigatus, N. fischeri, A. nidulans, A. niger, and A. kawachii incorporate the C before the template boundary into their templates. Superscripts to the right of the telomeric sequences indicate the manner in which the telomeric repeats were determined or predicted: ¹Proposed by our lab based on template sequence; ²Proposed by researchers based on telomeric sequence (<i>A. clavatus</i> from the fungal genome database at Broad Institute, <a href="http://www.broadinstitute.org/science/data#" target="_blank">http://www.broadinstitute.org/science/data#</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Chang1" target="_blank">[56]</a>, <i>A. fumigatus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Nierman1" target="_blank">[40]</a>, and <i>A. flavus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Chang2" target="_blank">[65]</a>); ³Identified by Bal31digestion and southern blot (<i>A. nidulans </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Bhattacharyya1" target="_blank">[22]</a>, <i>A. oryzae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058661#pone.0058661-Kusumoto1" target="_blank">[23]</a>); ⁴Proposed by our lab based on genome sequence data.</p

Strongly syntenous regions surround nine of the filamentous fungi TERs.

<p>The region to the right of TER shows more conservation of genes than the region to the left of TER. Chromosome VIII of A. nidulans contains TER, but no synteny is exhibited with these nine fungi, although five of the syntenous proteins were found dispersed across chromosome VIII of A. nidulans: vacuolar protein sorting protein, 3-oxoacyl-acyl carrier protein reductase, peptidyl-prolyl cis-trans isomerase, arrestin.</p