Absence of Rei1 affects the 60S maturation. (A) The rei1Δ strain displays an altered polysome profile. The disrupted strain and wild-type strain were grown for 16 h at 23°C in yeast extract–peptone–d-glucose (YPD). Whole cell extracts were separated on sucrose gradients as described in . In the rei1Δ strain (right), the position of half-mers is highlighted by asterisks. (B) Schematic representation of the maturation of the large subunit rRNA from 27SA2 to 5.8S and 25S. The positions of the oligonucleotide probes used in this study are indicated. (C) Effects of rei1Δ on pre-rRNA processing are similar to those of a TIF6 repression. Total RNAs were extracted from a rei1Δ or wild-type strain grown for 16 h at 23°C in YPD or from a PGAL1-TIF6 strain shifted to YPD from 0 to 6 h. 25S and 18S rRNAs were detected by ethidium bromide staining. 27SA2 and 27SB pre-rRNAs were detected by primer extension with probe CS10 (see ). 7S pre-rRNA, 5.8S rRNA, and total 27S species were detected by Northern blotting with the CS3, CS5, and CS10 probes, respectively. (D) The absence of Rei1 or Tif6 correlates with a defect in the C1 + C2 rRNA processing step. Ratios were calculated either between two mature rRNA or pre-rRNA detected in the same samples on the same gel or after normalization with a control RNA (U2 small nuclear RNA) detected with oligonucleotide MFR457. RNA levels in the rei1Δ strain were standardized to the wild-type levels, indicated as a dotted line; for the TIF6 repression, the levels were indicated relative to those at time = 0. For rei1Δ, the calculation was performed on four distinct experiments; error bars represent SD. (E) Rei1 is not a shuttling factor. Strains producing Rei1-GFP, Rpl25-GFP, Rlp24-TAP, or Rpl24b-TAP were transformed with plasmids pAJ544 and pAJ368 overexpressing wild-type NMD3 or the dominant-negative nmd3Δ100 allele, respectively. The TAP fusion proteins were revealed by immunocytochemistry, and all samples were observed by epifluorescence microscopy.
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