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. 2016 Nov 28;45(4):1731–1742. doi: 10.1093/nar/gkw1145

Figure 7.

Figure 7.

U1 snRNP interaction with the major late first leader 5΄ splice site has a suppressive effect on L4-22K-activated MLP transcription. (A) 5 μg total RNA from HEK293 cells cotransfected with 1.5 μg of luciferase reporter plasmid MLP, MLP12S or MLP12S-5΄ssM and activator plasmid pcDNA3-L4-22K (0.1 or 0.2 μg) was assayed by the primer extension technique. Cells were additionally cotransfected with 0.2 μg of pUC-U1-4u plasmid (lanes 13 and 14). ‘–’, denotes no L4-22K activator plasmid DNA added. The arrow points to the full-length primer extension product. (B) In vitro run-off transcription, 0.1 μg MLP template DNA linearized by NcoI cleavage was incubated with the recombinant L4-22K protein (0.2 or 0.5 μg) in 5S rRNA (lanes 1–3) or U1 snRNA depleted HeLa–NE (lanes 4–6). The MLP full-length transcript is 226 nucleotides in length. ‘–’ denotes no recombinant L4-22K protein added. The experiments shown in panels A and B have been conducted three and four times, respectively.