Fig 6.

Reduced poly(A) tail length affects L1 RNP formation. (A) Diagram of an L1 retrotransposition construct with a hammerhead ribozyme-aptamer controlled by theophylline. A self-cleavage ribozyme-aptamer (74) was inserted between the eGFPAI marker and the simian virus 40 (SV40) poly(A) signal that normally specifies polyadenylation in this construct. Upon adding theophylline to the medium, the ribozyme cleaves off the SV40 poly(A) signal and therefore leaves only a short oligo(A) tail (A₁₀). Orange circle, theophylline; TRE, tetracycline-regulated promoter; FLAG, FLAG epitope tag at C terminus of ORF1p; pLD289, L1 construct without the ribozyme cassette inserted; pLD437, L1 construct with the ribozyme cassette inserted. (B) Retrotransposition ability of pLD289 and pLD437 in HEK293T-tet-on cells with different concentrations of theophylline. The absolute activity of pLD289 (∼8%) was set at 1. (C) Relative mRNA levels of L1 ORF1 and ORF2 in Tet-on HEK293T cells (transfected with pLD437) with various concentrations of theophylline. Tet-on HEK293T cells were transfected with pLD437. The next day, doxycycline (final concentration, 500 ng/ml) and theophylline were added to the medium. At 5 days posttransfection, total RNA was prepared and real-time RT-PCR was performed. Both the beta-actin and puromycin genes were used as internal controls. All data represent averaged results for at least three independent experiments. Error bars represent standard deviations. (D) A shorter poly(A) tail affects L1 RNP formation. Tet-on HEK293T cells were transfected with pLD289 and pLD437. Transfected cells were selected in DMEM containing 1 μg/ml puromycin and 500 ng/ml doxycycline for 5 days. Theophylline was added to a final concentration of 10 mM and incubated for 2 more days. L1 RNPs were prepared from each cell pool and probed with anti-FLAG antibody. The signals were quantified using Multi-Gauge software (Fujifilm), based on band densitometry. For each sample pair, the signal with theophylline was compared to the signal without theophylline (assigned as 1), with normalization to signals from the total cell lysate (upper panel). The experiment was done twice from two independent transfections and cell cultures.