tRNA 3' processing in yeast involves tRNase Z, Rex1, and Rrp6

Abstract

Mature tRNA 3' ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3' endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3' processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3'-end processing of precursors with long 3' trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3' ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle.

Keywords: 3′-end processing; Saccharomyces cerevisiae; exonucleases; tRNA; tRNase Z.

FIGURE 1.

Trz1-TAP is localized in both nucleus and mitochondria. (A) Subcellular fractions of yeast extracts (total cells, cytosol, mitochondria, and nuclei) were analyzed by Western blotting using peroxidase-anti-peroxidase antibodies (PAP). The same blot was probed against nuclear (Nop1), mitochondrial (Mdh1), and cytoplasmic (Hxk2) proteins using specific antibodies. Ponceau S staining is shown in the bottom panel as a loading control. (B) Localization of Trz1-TAP in yeast mitochondrial matrix. Alkali-extracted soluble and membranes fractions as well as total mitochondrial extract were analyzed by Western blotting using PAP antibodies for Trz1 and specific antibodies for markers of different mitochondrial compartments: Mdh1 (matrix, M), Tim23 (inner membrane, IM) and Tom70 (outer membrane, OM). Asterisks mark bands resulting from antibodies’ cross-reactivity.

FIGURE 2.

Yeast tRNA precursor is efficiently processed by recombinant Trz1. (A) Processing of an in vitro transcribed yeast nuclear pre-tRNA^Ser by 100 ng of recombinant Trz1. Lane p, processing reaction; lane c, control reaction without the protein. Precursor and products (mature tRNA and a 3′ trailer) are shown schematically on the right; dashed line denotes the intron in tRNA^Ser; DNA size marker (lane M, in nt) is shown on the left. (B) Determination of Trz1 cleavage site in vitro on pre-tRNA^Ser by primer extension. The major cleavage after the discriminator base and two additional, minor cleavages are indicated with arrows. A processing reaction was carried out with nonlabeled pre-tRNA, and the resulting 3′-trailer product was isolated and used as a template in the primer extension reaction. Sequencing reaction (lanes G,A,T,C) performed with the same primer (YC1) is shown on the left. The location of the primer relative to the tRNA is illustrated schematically at the bottom.

FIGURE 3.

Processing of tRNA precursors with long 3′ trailers is affected in trz1 mutants. (A) Growth curves of wild-type (BMA38, ⧫) and GAL::HA-Trz1 (▪) strains pregrown in YPGal medium (permissive conditions) and shifted to YPD medium (nonpermissive conditions) for the times indicated. Strains were maintained in exponential growth by dilution with fresh medium. Cell densities measured by optical density at 600 nm are shown corrected for dilution. (B) Depletion of Trz1 in the GAL::HA-Trz1 strain grown as described in A; the wild-type strain grown in YPD is shown as a control. Western blotting for proteins extracts prepared from cells harvested at indicated time-points following depletion was probed with anti-HA antibodies. Equal amounts of total protein were loaded in each lane. (C) Potential stem–loop structures in the 3′ extensions of pre-tRNAs with long 3′ trailers. (D,F) Northern analysis of tRNA processing for precursors with long 3′ trailers in the GAL::HA-Trz1 (D) and trz1-3 (F) strains. GAL::HA-Trz1 cells were grown as described in A; trz1-3 cells were pregrown at 25°C (0 h) and transferred to 37°C for the times indicated. RNA was separated on an 8% polyacrylamide gel and hybridized with oligonucleotide probes. Probe names are indicated in parentheses. tRNA species and their graphic representations, with the position of probes used for hybridization, are shown on the right. Asterisk marks cross-hybridization to another RNA. Mature tRNAs are not shown, as specific products of analyzed precursors cannot be detected, and the probes against mature species hybridize to all tRNA^Lys1 or tRNA^Phe molecules. (E) Temperature-sensitive phenotype of the trz1-3 mutant. Wild-type and trz1-3 strains were grown on YPS plates at 30°C and 37°C.

FIGURE 4.

Both Trz1 and Rex1 contribute to tRNA 3′ processing. (A) Growth curves of GAL::HA-Trz1 (▪), GAL::HA-Trz1/rex1Δ (▴), GAL::HA-Trz1/rrp6Δ (□), GAL::HA-Trz1/rex2Δ/rrp6Δ (•), and GAL::HA-Trz1/trf4Δ/rex1Δ (○). Description is as in Figure 3A. Strains with overlapping curves (GAL::HA-Trz1, GAL::HA-Trz1/rex1Δ, and GAL::HA-Trz1/trf4Δ/rex1Δ or GAL::HA-Trz1/rrp6Δ and GAL::HA-Trz1/rex2Δ/rrp6Δ) exhibit similar growth rates. (B–D) Northern analysis of tRNA processing in mutants lacking different combinations of nucleases: strains deleted for Rex1-3 and Rrp6 (B); GAL::HA-Trz1 and GAL::HA-Trz1/rex1Δ strains (C); GAL::HA-Trz1/rex2Δ/rrp6Δ strains (D). Respective wild-type (WT) strains were used as controls. GAL::HA-Trz1 strains were grown as described in Figure 3. Probe names are indicated in parentheses; tRNA species with the position of probes used for hybridization are shown on the right. Asterisks in B denote 3′-ptRNA^Ile1 in rex1Δ mutants, that has a similar migration as 5′/3′-ptRNA^Ile1 in other strains; diamonds and triangles in B and C mark extended and standard 5′/3′- ptRNA^Ile1 or 5′/3′-ptRNA^Lys2 that have different migration in rex1Δ and other strains.

FIGURE 5.

tRNA precursors with long 3′ trailers are markedly affected and polyadenylated by Trf4 in cells lacking both 3′ processing activities. (A,B) Northern analysis of tRNA_VII ^Phe and tRNA_IV ^Lys1 in wild-type, GAL::HA-Trz1, rrp6Δ and GAL::HA-Trz1/rrp6Δ (A) or wild-type, GAL::HA-Trz1, rex1Δ and GAL::HA-Trz1/rex1Δ strains (B). (C) Pre-tRNA_VII ^Phe are polyadenylated in GAL::HA-Trz1/rex1Δ cells. Northern hybridization for tRNA_VII ^Phe of total RNA (lanes 1–4) from GAL::HA-Trz1/rex1Δ mutant (m samples) treated with RNase H in the absence (−) and presence (+) of oligo(dT); or of the total or poly(A)⁺ RNA fraction (A⁺) (lanes 5–8). Respective wild-type (WT) strains were used as controls. (D) Polyadenylation of tRNA_VII ^Phe in the GAL::HA-Trz1/rex1Δ strain is carried out by Trf4. Northern analysis of tRNA_VII ^Phe in wild-type, GAL::HA-Trz1, trf4Δ/rex1Δ, GAL::HA-Trz1/rex1Δ and GAL::HA-Trz1/trf4Δ/rex1Δ strains. GAL::HA-Trz1 strains were grown as described in Figure 3. Probe names are indicated in parentheses. tRNA species with the position of probes used for hybridization are shown on the right. Diamonds and triangles in A, B, and D denote 5′/3′-ptRNA_VII ^Phe that have different migration in wild-type, rex1Δ and rrp6Δ strains; asterisk marks cross-hybridization to another RNA.

FIGURE 6.

Synthesis of mitochondrial transcripts depends on Trz1. (A) trz1 mutants are respiratory-deficient. Wild-type (WT), GAL::HA-Trz1, and trz1-3 strains were grown on plates containing glycerol as a sole carbon source. (B) DAPI staining to visualize nuclear and mitochondrial DNA in wild-type (WT), GAL::HA-Trz1, rho⁻, and rho0 cells by fluorescent microscopy. (C) Northern analysis of mature mt-tRNAs in the mitochondrial RNA fraction of wild-type (WT) and GAL::HA-Trz1 strains grown at permissive conditions (galactose). (D) Northern analysis of tRNA^fMet-RPM1-tRNA^Pro and tRNA^Glu-COB1 mitochondrial transcription units using total RNA of wild-type (WT) and GAL::HA-Trz1 strains grown at permissive conditions. Probe names are indicated in parentheses, identities of RNA transcripts are shown on the right, graphic representations of corresponding mitochondrial transcription units as in Tzagoloff and Myers (1986); Foury et al. (1998); Schonauer et al. (2008), with the position of probes used for hybridization, are depicted on the left (C) or on the right (D). RNAs marked with diamonds represent aberrant RPM1 precursors or processing intermediates. Promoters are depicted as flags. SCR1 was used as a loading control. Note that the mature tRNA^fMet in the tRNA^fMet-RPM1-tRNA^Pro unit can originate from two precursors, pre-tRNA^fMet or tRNA^fMet-RPM1-tRNA^Pro (Stribinskis et al. 1996).

FIGURE 7.

Mitochondrial nucleases contribute to the overall processing of mtRNAs. Northern analysis of tRNA^fMet-RPM1-tRNA^Pro transcription units using total RNA of wild-type, rex2Δ, nuc1Δ, pet127Δ, GAL::HA-Trz1 single-mutants and GAL::HA-Trz1/rex2Δ, GAL::HA-Trz1/nuc1Δ, and GAL::HA-Trz1/pet127Δ double-mutants. Corresponding wild-type strains, NB80 for pet127Δ mutants and YRP840 for the remaining mutants, were used as controls. Probe names are indicated in parentheses, identities of RNA transcripts are shown on the right. Graphic representation of the tRNA^fMet-RPM1-tRNA^Pro transcription unit and major detected precursors and intermediates is shown below. SCR1 was used as a loading control.