3'-processing of yeast tRNATrp precedes 5'-processing - PubMed (original) (raw)
3'-processing of yeast tRNATrp precedes 5'-processing
Joanna Kufel et al. RNA. 2003 Feb.
Abstract
Previous analyses of eukaryotic pre-tRNAs processing have reported that 5'-cleavage by RNase P precedes 3'-maturation. Here we report that in contrast to all other yeast tRNAs analyzed to date, tRNA(Trp) undergoes 3'-maturation prior to 5'-cleavage. Despite its unusual processing pathway, pre-tRNA(Trp) resembles other pre-tRNAs, showing dependence on the essential Lsm proteins for normal processing and efficient association with the yeast La homolog, Lhp1p. tRNA(Trp) is also unusual in not requiring Lhp1p for 3' processing and stability. However, other Lhp1p-independent tRNAs, tRNA(2)(Lys) and tRNA(1)(Ile), follow the normal pathway of 5'-processing prior to 3-processing.
Figures
FIGURE 1.
Processing of pre-tRNATrp. (A_–_T) RNA was extracted from wild-type cells grown at 23°C (lane 1) or 12 h after transfer to 37°C (lane 2); from the otherwise isogenic rpr1-1 strain grown at 23°C (lane 3) or after transfer to 37°C for 2, 6, 10, and 22 h (lanes 4_–_7); and from otherwise isogenic wild-type and _lhp1_-Δ strains grown at 30°C (lanes 8,9). Names of tRNAs are on the left, schematic representations of tRNA precursors, intermediates, and mature species are shown on the right. Probe names are shown in parentheses, and their locations are indicated as lines under the schematics of the pre-tRNA species and in panel U. (V) Sequences immediately downstream of the 3′-mature ends of the five pre-tRNATrp species.
FIGURE 2.
Normal processing of pre-tRNATrp requires Lsm proteins. Processing of pre-tRNATrp and pre-tRNA3Leu in GAL-lsm (A) and _lsm_ts (B) strains. Strains carrying _GAL_-regulated constructs (GAL|mBlsm, A, lanes 3_–_8) and the BMA64 wild-type strain (WT, A, lanes 1,2) were grown in permissive RSG medium (0 h) and transferred to repressive glucose medium at 30° for the times indicated. The temperature-sensitive _lsm2_ts and _lsm5_ts strains (B, lanes 3_–_8) and the wild-type strain (WT, A, lanes 1,2) were pregrown at 23°C (0 h) and transferred to 37°C for the times indicated. RNA was separated on a 6% polyacrylamide gel and hybridized with oligonucleotide probes.
FIGURE 3.
Efficient association of pre-tRNATrp with Lhp1p requires Lsm3p. (A) Effects of Lsm3p depletion on the association of Lhp1p with pre-tRNATrp and pre-tRNA3Leu. Lysates were prepared from strains GAL|mBlsm3 (lanes 1_–_3), Lhp1p-ProtA (lanes 4_–_6), and Lhp1p-ProtA; GAL-lsm3 (lanes 7_–_12) after being grown at 30°C in RSG medium (lanes 1_–_9) or after transfer to glucose medium for 24 h (lanes 10_–_12) and immunoprecipitated using IgG agarose. RNA was recovered from the total lysate (T), immune supernatant (S), and the immune pellet (P) and was analyzed by Northern hybridization. Approximately fourfold more cell equivalents are loaded for the pellet fraction. The RNA band indicated with an asterisk results from degradation during immunoprecipitation. (B) Graphic representation of the immunoprecipitation efficiency of RNAs in the Lhp1-ProtA/GAL-lsm3 strain in RSG medium (0 h, white bars) and after transfer to glucose medium for 8.5 h (hatched bars) and 24 h (black bars). Values for each RNA species after depletion are expressed relative to the value before depletion, which is arbitrarily set at one.
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