Abstract
The 3′ end of Schizosaccharomyces pombe telomerase RNA (SpTER1) is generated by spliceosomal cleavage, a reaction that corresponds to the first step of splicing. The observation that the spliceosome functions in 3′ end processing raised questions about the evolutionary origin and conservation of this mechanism. We now present data in support of spliceosomes generating 3′ ends of telomerase RNAs in other fungi. Strikingly, the mechanistic basis for restricting spliceosomal splicing to the first transesterification reaction differs substantially among species. Unlike S. pombe, two other fission yeasts rely on hyperstabilization of the U6 snRNA—5′ splice site interaction to impede the 2nd step of splicing. In contrast, a non-canonical 5′ splice site blocks the second transesterification reaction in Aspergillus species. These results demonstrate a conserved role for spliceosomes functioning in 3′ end processing. Divergent mechanisms of uncoupling the two steps of splicing argue for multiple origins of this pathway.
Highlights
The 30 end of Schizosaccharomyces pombe telomerase RNA (SpTER1) is generated by spliceosomal cleavage, a reaction that corresponds to the first step of splicing
To examine whether the mechanism of telomerase RNA 30 end processing is conserved across fission yeast species, we identified the telomerase RNA subunits from S. cryophilus and S. octosporus based on synteny in the corresponding regions of the genomes followed by alignment with SpTER1
The template region, which is generally more highly conserved among telomerase RNAs, differs between S. pombe and the other fission yeast species, with S. cryophilus and S. octosporus sequences containing a three-nucleotide insertion relative to S. pombe (Supplementary Fig. 1). When this sequence was inserted into S. pombe TER1 and telomeres were cloned and sequenced, near-perfect GGGTTACTT repeats were added to chromosome ends rather than the heterogeneous G2–6TTACA0–1C0–1 repeat generated by the endogenous template (Supplementary Fig. 2)
Summary
The 30 end of Schizosaccharomyces pombe telomerase RNA (SpTER1) is generated by spliceosomal cleavage, a reaction that corresponds to the first step of splicing. A non-canonical 50 splice site blocks the second transesterification reaction in Aspergillus species These results demonstrate a conserved role for spliceosomes functioning in 30 end processing. These features attenuate the transition from first- to second-step conformation and result in the release of the splicing intermediates This process is akin to ‘discard’, a pathway that has been characterized through a series of elegant experiments in budding yeast where the DExD/H-box helicase Prp[22] functions in the recovery of spliceosomes that have selected suboptimal 30 splice sites[14,15]. While the use of the first step of splicing to generate telomerase RNA 30 ends is conserved, the RNA elements that uncouple the two steps of splicing are entirely different
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