Antisense RNA oligonucleotides to spliceosomal snRNAs, e.g. masking those regions of U2 or U12 snRNAs complementary to the intron branch site, can selectively interfere with assembly of the two splicing machineries in in vitro
splicing reactions using nuclear extracts and synthetic RNA substrates (7
). For a strategy to selectively target spliceosomal snRNAs in living cells, we decided to use non-ionic antisense morpholino oligomers (9
). Morpholinos bind with high specificity to transcripts and have been used to block splice sites in pre-mRNA (10
) and mRNA translation (12
) in vivo
. To selectively interfere with U12- and U2-type splicing, we chose morpholinos complementary to sequences in U12 and U2 snRNA base-pairing with the distinct branch sites of the two splicing systems ().
Figure 1 Schematic representation of morpholino-mediated targeting of U12 (A) and U2 snRNA (B). U12 and U2 snRNPs with snRNA sequences base-pairing with the corresponding branch point consensus sequence (19) in pre-mRNA are shown (R, purine; Y, pyrimidine; and (more ...)
In studies using lymphoma cells, we recently found that plain morpholinos, although being non-ionic, can be efficiently and cheaply transfected by electroporation, even into cells for which electroporation of plasmid DNA is much less efficient [(14
); and our unpublished data)]. To test the potential of our morpholino oligomers to interfere with either splicing apparatus in vivo
, they were electroporated into mouse EL4 T-lymphoma cells together with a four-exon minigene construct derived from the human P120
) (), containing U2-dependent introns (E and G) and a U12-type intron (F). Splicing of introns F (U12-type) and G (U2-type) from the minigene RNA was then monitored after RT of total cellular RNA and PCR using primers (see ) hybridizing in the upstream and downstream exons, respectively. Intron F removal, reflected by the amplification product lacking the intron, was inhibited with increasing amounts of the antisense-U12 oligomer (u12MO) co-transfected (, lanes 2–7). A non-specific control morpholino (cMO, lane 8) and the antisense-U2 sequence (u2MO, lane 9), in a concentration inhibiting U2-type splicing (cf. , lane 6), had no effect. Conversely, increasing doses of the u2MO sequence interfered with splicing of U2-dependent intron G (, lanes 2–6), while the control morpholino and the antisense-U12 morpholino in the highest amount did not affect the removal of the intron (lanes 7 and 8). Inhibition of U2-type splicing, however, required several-fold higher morpholino doses (), which could be caused by differences in the efficiency of morpholino hybridization or reflect the high abundance of U2 snRNP relative to U12 snRNP (16
). The low abundance of the U12-type spliceosome, together with the high levels of minigene expression, may also explain the incomplete splicing of intron F from the minigene transcripts (, lane 2) relative to endogenous P120 pre-mRNA (, upper panel, lane 2; the same result was also obtained in the EL4 cells, data not shown). In conclusion, the findings indicate selective inhibition of U2- and U12-type splicing by delivering the corresponding antisense-morpholino sequences to cells.
Figure 2 Selective inhibition of U2- and U12-type splicing in vivo. (A) Schematic representation of human P120 minigene (15) with promoter (open arrow), exons (open boxes) and U2- and U12-type intron sequences (E–G, bold lines). Arrowheads indicate positions (more ...)
Figure 3 Inhibition of U12-type splicing from endogenous genes. (A) Antisense-U12 morpholino-mediated inhibition of splicing at different time points after morpholino transfection. Human Jurkat-leukemia cells were transfected with 15 nmol of the u12MO or of a (more ...)
To test the suitability of the method for analyzing splicing of endogenous pre-mRNAs, we delivered the antisense-U12 oligomer to the commonly used human Jurkat-leukemia cell line by electroporation and monitored intron F removal from the human P120 pre-mRNA at different time points after morpholino transfection (, upper panel). While intron removal was complete immediately after transfection (lane 2), transcripts without intron F became almost undetectable after 6–12 h; instead, the intron-containing RNA accumulated (lanes 2–4), indicating the suppression of intron F splicing. The non-specific control sequence (lane 6) and the antisense-u2MO oligomer (not shown in this panel, but see ) had no effect. P120 intron F represents U12-type introns having AT and AC dinucleotides at their 5′ and 3′ boundaries, respectively (15
). As an example for an U12-type intron with GT and AG terminal nucleotides, we examined splicing of intron 22 of the human poly-ADP-ribosyl transferase (ADPRT
) gene (17
) (, lower panel). Again, transfection of the antisense-U12 morpholino blocked original complete splicing of the intron (lane 2), and led to the accumulation of the unspliced transcript over a period of 18 h (lanes 2–5). The suppression of U12-type splicing by the u12MO oligomer from both endogenous genes occurred in a dose-dependent manner (, lanes 2–8). Specificity is indicated by the non-specific control morpholino (lane 9) and the antisense-U2 sequence (lane 10), which did not affect intron removal. These results indicate that the approach is effective enough for restraining spliceosome function on endogenous transcripts. At the same time, they establish a role for U12 snRNA in the splicing of endogenous pre-mRNAs.