Figure 5.

L4-22K recruits U1 snRNP to the major late leader 1 exon. (A) RNA from HeLa–NE pretreated with RNase H and a complementary oligonucleotide directed against the 5΄-end of U1 snRNA (U1 oligo) or the 5S rRNA (5S oligo) was separated on an 8% denaturing polyacrylamide gel. The position of the full length (U1) and shortened (U1*) U1 snRNA are indicated. (B) 0.2 μg recombinant L4-22K protein was incubated with 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (depicted in panel E) and 1.5 μg U1 snRNA-depleted HeLa–NE (U1 oligo) or 5S rRNA-depleted HeLa–NE (5S oligo) in a gel shift assay. (C and D) 0.4 μg recombinant L4-22K protein was incubated with 2.6 μg HeLa–NE and 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (WT in panel C and D), the sense-strand major late first leader-R1 5΄splice site mutant ssRNA (5΄ssM in panel C) or the sense-strand major late first leader-R1 5΄-CAAA-3΄ motif mutant ssRNA (MR4 in panel D). ‘–’ denotes no recombinant L4-22K protein added. (E) Schematic drawing depicting the structure of the major late first leader-R1 ssRNA used in the gel shift assays. (F) The sequence of the R1 wild type, mutant MR4 and the 5΄ splice site mutant sense-strand ssRNAs used as probes in the gel shift assays. (G) HEK293 cells were co-transfected with a Flag-L4-22K expressing plasmid and an MLP reporter plasmid lacking the DE element. Following a 36 hours incubation extracts were UV crosslinked and immunoprecipitated with an anti-Flag or anti-IgG antibodies, RNA was extracted and quantitated by RT-qPCR. Results are shown as the fold change of the major late first leader-R1 ssRNA (MLT) or U1 snRNA in Flag-IP versus IgG-IP. (H) Immunoblotting of the same samples used in panel G using antibodies directed against Flag-L4-22K and Actin. The experiments shown in panels B and D have been conducted three times and in panel C four times. The data shown in panel G is the result from three independent experiments with error bars indicating the standard deviation.