Figure 3.
HAC1 intron is circularized and retained in the AtRLG1 strain. Wild-type (RLG1), rlg1-4, rlg1-100, ire1Δ, RLG1-HA, and rlg1Δ/HA-AtRLG1[M74] strains were cultured in the presence (+ lanes) or absence (− lanes) of 2.0 μg/ml Tm for 60 min. Total RNAs were prepared and electrophoresed with 1.5% agarose/2.2 M formaldehyde gel. (A) HAC1 RNA species were detected by Northern blotting with an anti-HAC1u probe (top). ACT1 mRNA was also detected as a loading control (bottom). (B) The total RNAs from the AtRLG1[M74] cells were subjected to Northern blotting with an anti-HAC1 intron probe. Signal enhancement enables to observe the minor HAC1u mRNA in the presence of Tm. (C) Total RNAs prepared from HA-AtRLG1 cells were hybridized with probes a or b and treated with RNase H. In the lane labeled −, no oligonucleotide was added. RNAs recovered from the digestion were analyzed on a polyacrylamide gel. The HAC1 intron and its fragments were visualized by Northern blotting with either of the probe a (left) or b (right). Hybridization positions of these probes on putative linear or circular HAC1 introns are schematically described in the right.