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. Author manuscript; available in PMC: 2009 Nov 12.
Published in final edited form as: Nat Struct Mol Biol. 2009 Feb 8;16(3):255–264. doi: 10.1038/nsmb.1556

Figure 5.

Figure 5

The proximal (non-canonical) polyadenylation signal regulates splicing and accumulation of crs1 RNA. a, Schematic diagrams indicating the regions interchanged in nmt81-crs1 chimerae, which are demarcated by brackets and designated by numbers. b, RT-PCR splicing assays on nmt81-crs1 chimerae. The letters above each lane on the gel indicate the source of the segments flanking the ORF, which correspond to the numbers on the schematic, with a dash representing the coding region. Heterologous flanking segments were derived from a thiamine-responsive expression plasmid in which the inserted ORF is surrounded by the nmt81 transcriptional and translational control regions40,41. Analyses were carried out in the crs1 deletion strain JLP9702 to ensure that all products were derived from the plasmid-borne alleles. c, Splicing assays on crs1 polyadenylation signal mutants. Lane 1: wild-type control. Lane 2: NN-CN construct (parent for the remaining alleles). Lanes 3 and 4: either the proximal (non-canonical) or distal (canonical) 57 nt polyadenylation signal (Fig. 2b) was replaced with complementary sequences. d, qPCR analysis of spliced and unspliced crs1 in the same RNA samples analyzed in Panels a-c, plus two additional chimerae (9 & 10). The percentage of spliced crs1 RNA is indicated next to each allele designation.