A′ cleavage can be bypassed. (A) HeLa cells were transfected with control siRNAs or those targeting XRN2 or RRP6, either individually or together. Sixty hours later, RNA was harvested and analyzed by Northern blotting using probes hybridizing between the 5′ end of the pre-rRNA transcript and A′ (ETS1) or A′-A0 (ETS2). Mature rRNAs were visualized by methylene blue staining. (B) The experiment was repeated using siRNAs targeting MTR4 instead of RRP6. Note that the 37S* band seen with the XRN2 knockdown was not consistently observed. (C) RNA was extracted from various different cell lines and after differentiation for TC7 cells. This was analyzed by glyoxal gel electrophoresis, followed by Northern blotting using a probe hybridizing upstream of the A′ cleavage site (ETS1).
