Diverse mechanisms for spliceosome-mediated 3' end processing of telomerase RNA

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.

Figure 1. Identification and characterization of telomerase RNA from Schizosaccharomyces cryophilus and S. octosporus.

(a) Northern blot for telomerase RNA from S. cryophilus (left) and S. octosporus (right). Total RNA of the respective species was separated on a 4% polyacrylamide gel and probed with ³²P-labelled fragments corresponding to the first exon of the respective TER1 gene. (b) RT–PCR using primer pairs spanning the putative introns in ScTER1 and SoTER1. The identities of the upper band as unspliced and lower band as spliced products were verified by sequencing. (c) Schematic of the introns located downstream of the 3′ ends of the mature forms in each of the three species. Distances between the Sm-binding site, 5′ splice site, branch site and 3′ splice site are given in nucleotides (nts). Designation of the Sm-/LSm-binding sites in S. cryophilus and S. octosporus is based on sequence similarity, not on experimental verification of Sm/LSm binding. To indicate this fact, the sites are labelled with a question mark. (d) RNaseH cleavage followed by northern blot visualizes the relative abundance of precursor, spliced and cleaved forms of SpTER1 containing the respective introns (colours as in c). An oligonucleotide probe against the snoRNA sn101 was used as a loading control.

Figure 2. Hyperstabilization of the 5′SS:U6 snRNA interaction impedes completion of splicing for the introns from S. cryophilus and S. octosporus.

(a) Schematic of interactions between the 5′SS found in S. octosporus and S. cryophilus TER1 and U1 and U6 snRNA, respectively. The single-nucleotide change C3 to A (indicated in green) adds an A:U interaction for U1, but destabilizes the interaction with U6 snRNA. (b) RNaseH cleavage followed by northern blot to visualize precursor, cleaved and spliced forms of TER1 containing the intron from S. pombe (green), S. cryophilus (blue) and S. octosporus (red) or mutant versions thereof. (c) RT–PCR visualizing relative abundance of precursor and spliced forms for the different constructs. LC, loading control.

Figure 3. A non-canonical 5′ splice site permits spliceosomal cleavage during telomerase RNA biogenesis in filamentous fungi.

(a) Partial alignment of telomerase RNAs from five Aspergillus species and Neosartorya fischeri generated in ClustalW2. Asterisks mark positions conserved across all six species. The conserved 5′SS, branch site and 3′SS are highlighted in blue, green and grey, respectively. (b) Schematic of constructs used to examine processing of A. niger sequences (orange) in the context of SpTER1 (green). (c) Untreated and poly(A) polymerase (PAP)-treated total RNA was subjected to reverse transcription in the presence of oligo dT followed by PCR to clone the 3′ ends of naturally polyadenylated (minus PAP lanes) and non-polyadenylated forms (product only observed in +PAP lanes). (d) Position of 3′ ends of the non-polyadenylated (cleaved) form of TER1 containing the intron and downstream sequence from A. niger.

Figure 4. The non-canonical 5′ splice site blocks completion of splicing and promotes spliceosomal cleavage of the A. niger TER1 intron.

(a) Northern blot on total RNA from cells expressing the wild-type A. niger intron or the A1 to G mutation shown in red. (b) RT–PCR visualizing precursor and spliced forms. (c) Telomerase activity assay using extracts from cells expressing telomerase RNA containing wild-type or mutant versions of the introns from S. pombe or A. niger, respectively. Wild-type sequence of 5′SS shown in black, mutated nucleotides in red. (d) Telomere length determined by Southern blotting of EcoRI-digested genomic DNA. Vector denotes the absence of telomerase RNA (biological replicates in lanes 1 and 4, WT denotes the wild-type version of S. pombe TER1 (lane 3), AUAAGU denotes the wild-type version of the A. niger intron (biological replicates in lanes 2 and 5) and GUAAGU denotes a mutated 5′SS in the A. niger intron (biological replicates in lanes 6 and 7). A 300-bp telomeric DNA fragment was used as a template for nick-translation to generate a ³²P-labelled probe; a second probe for the rad16 gene was used as a loading control (LC).

Figure 5. The effects of non-canonical 5′ splice sites are context dependent.

(a) RT–PCR for the tif212 intron and 5′SS mutations in the context of TER1. (b) Northern blot for TER1 containing introns from protein-encoding genes with non-canonical 5′SS (GUCAGU, lanes 2–4; GUCUGU, lane 5). (c) Schematic of mechanisms that promote spliceosomal cleavage of telomerase RNA in different organisms. LC, loading control.