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).