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1.
FIGURE 2.

FIGURE 2. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

Stem length of anticodon stem–loop (ASL) substrates of Tit1p. (A) Predicted structures of ASL oligos representing tRNASer and tRNATyr of various lengths; A37 is circled. Nucleotide positions 28 and 33 are indicated for SerAGA-17; numbering is the same for all others. For some, an extra closing G-C base pair was added (lowercase). For TyrGUA-19, an A-U base pair was added for comparison to SerAGA-19. (B) In vitro modification of the ASLs in A using recombinant Tit1p and 14C-DMAPP. (C) SerAGA-19 (lane 1) and its derivative ASLs with the substitutions indicated above lanes 2–7 were assayed as in B.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
2.
FIGURE 3.

FIGURE 3. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

The Tit1p extended C terminus including Zn finger is critical for activity in vitro and in vivo. (A) Equal amounts of purified recombinant proteins were tested for in vitro activity using 14C-DMAPP and total RNA from yNB5 (tit1-Δ) as substrate, with protein(s) as indicated above the lanes. (B) The gel in A stained with ethidium bromide. (C) tRNA-mediated suppression assay of the strains transformed with the expression plasmids indicated. (D) Proteins from the yNB5 strains in C transformed with HA-tit1+ (lane 1), HA-tit1(1-379) (lane 2), and HA-tit1-ZnAA (lane 3) were examined by immunoblotting using anti-HA antibody.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
3.
FIGURE 1.

FIGURE 1. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

Deletion of tit1+ causes loss of tRNA i6A and tRNA-mediated suppression. Various strains growing in EMM with nonlimiting (A) or limiting (B) adenine, the latter reflecting tRNA-mediated suppression (TMS) activity. yYH1 (WT, tit1+), the parent strain of yNB5, was transformed with empty vector (row 1); this strain exhibits partial TMS activity (Huang et al. 2005). yNB5 (tit1-Δ) was transformed with empty vector, tit1+, tit1-T12A, or MOD5, as indicated for rows 2–5. (C,D) Analysis of S. pombe strains in A and B for i6A in tRNA by mid-Western blotting (Benko et al. 2000). (C) Ethidium bromide–stained total RNA in TBE-urea gel. (D) Blot of C after incubation with anti-i6A antibody and secondary processing for chemiluminescence (only the tRNA region is shown). (E) The tit1+ (lane 1), tit1-T12A (lane 2), and empty vector (lane 3) transformed strains from above were analyzed by immunoblotting using anti-HA antibody.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
4.
FIGURE 7.

FIGURE 7. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

The anticodon binding loop of Mod5p alters tRNA substrate preference. (A) In vitro modification of S. cerevisiae mod5-Δ RNA by wild-type Mod5p (lane 1) and various mutated Mod5 proteins (lanes 2–5). Mod5p-loop+K127D (lane 2) contains an 8-amino-acid insertion after position 120 as well as the K127D mutation; the Mod5 proteins used in lanes 3–5 are single point mutations as described above the lanes. (B) Quantitative scanning of each lane of the gel shown in A using a Fuji PhosphorImager. For normalization, the total counts observed in tRNAsSer in Mod5p (wild-type, lane 1) were considered as 100%, and the others were compared accordingly. Note that the black tracing (Mod5p-K127D) for tRNAsSer is not visible because it was completely masked (overlaid) by the red tracing (Mod5p wild type). (C) In vitro modification of ASL substrates indicated above the lanes by Mod5p wild type and Mod5p-loop+K127D; (upper panel) EtBr staining of gel autoradiogram shown in lower panel. (D) In vitro modification of S. cerevisiae MOD5 (ABL8) and mod5-Δ (MT8) RNA by purified recombinant human TRIT1.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
5.
FIGURE 5.

FIGURE 5. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

S. cerevisiae tRNATrp is not a substrate of Mod5p in vitro or in vivo. (A–E) PHA6 assay of S. cerevisiae RNAs from MOD5 replete and mod5-Δ cells as described in Figure 4 but using probes complementary to the ACL and TΨC regions of the S. cerevisiae tRNAs schematically depicted in F and indicated to the right; the same blot was probed, stripped, and reprobed sequentially. (G) In vitro modification by purified recombinant Tit1p, of S. cerevisiae tRNAs from mod5-Δ cells after elimination of specific tRNAs by RNase H, to identify in vivo substrates of MOD5. In vitro modification using Tit1p of RNA from ABL8 (MOD5) cells after mock preincubation (lane 1) or preincubation with oligo-DNA antisense to S. cerevisiae tRNATrp, both followed by RNase H treatment and purification prior to the 14C-DMAPP modification assay (lane 2). (Lane 3) Tit1p-mediated modification of S. cerevisiae RNA isolated from MT8 (mod5-Δ) after mock preincubation. (Lanes 4–10) RNA from MT8 (mod5-Δ) was preincubated with an oligo-DNA(s) antisense to the anticodon loop of the tRNA(s) targeted for elimination indicated above the lanes followed by RNase H and purification, prior to the Tit1p 14C-DMAPP modification assay. The tRNAs assigned to the bands are summarized to the right. (H) Comparison of Tit1p and Mod5p for in vitro modification of tRNAs from S. cerevisiae. (Lanes 1,2) ABL8 (MOD5) tRNA; (lanes 3,4) MT8 (mod5-Δ) tRNA modification by Tit1p and Mod5p. The tRNAs assigned to the bands are summarized to the right. (I) Ethidium-stained gel of the assay in H.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
6.
FIGURE 4.

FIGURE 4. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

Identification of tRNATrpCCA as a substrate of Tit1p. (A) Nineteen-nucleotide ASL oligos representing tRNATrpCCA from S. pombe and S. cerevisiae used in B for in vitro modification by Tit1p; SerAGA-19 (lane 1) from S. pombe is a control; Trp-19 from S. pombe (lane 2) and Trp-19 from S. cerevisiae (lane 3). The S. pombe and S. cerevisiae ASLs Trp-19 run differently on TBE-urea gel (see text). (C) In vitro modification assay after elimination of specific tRNAs by RNase H, to identify S. pombe substrates of Tit1p. (Lanes 1,2) In vitro modification by Tit1p of cellular RNA isolated from yYH1 (tit1+, lane 1) and yNB5 (tit1-Δ, lane 2). (Lanes 3–10) RNA from yNB5 was pre-incubated with oligo-DNA(s) complementary to the anticodon loop of the tRNA(s) targeted for elimination indicated above the lanes, followed by RNase H. The RNA was then purified prior to the Tit1p modification assay. (D–H) Positive hybridization in the absence of i6A37 (PHA6) assay to identify in vivo substrates of Tit1p in S. pombe. (D) The EtBr-stained gel from which the blot hybridizations below it were derived. Lanes 1–4 and 5–8 contain duplicate samples of 5 μg and 10 μg of RNA from yYH1 (tit1+) and yNB5 (tit1-Δ) as indicated. The same blot was probed, stripped, and reprobed sequentially with the probes indicated to the right. (E,G) The 32P-labeled probes were complementary to the anticodon loop (ACL) region of the tRNAs indicated to the right. (F,H) The 32P-labeled probes were complementary to the TΨC region of the tRNAs indicated to the right. The relative positions of the ACL and TΨC probes are indicated below the blots.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.
7.
FIGURE 6.

FIGURE 6. From: Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases.

C34G substitution activates tRNATrpCCA as a Mod5p substrate. (A) Comparison of the ASL regions of the two groups of natural Mod5p substrates and the nonsubstrate tRNATrpCCA, all of which contain A36A37A38. (Upper row) The three tRNAsSer substrates that comprise the non-G34 group and contain G35; (lower row) the first two substrates represent the G34 group, composed of tRNATyrGUA and tRNACysGCA; (lower right) the nonsubstrate tRNATrpCCA. (B–D) In vitro modification of ASLs: (B) Mutations to the stem of ASLs representing tRNATrpCCA do not activate it as a Mod5p substrate. (Lane 1) Ser-AGA-19; (lane 2) scTrp-19; (lane 3) scTrp-19-mut2-C29A•G41U; (lane 4) scTrp-19-mut6: U27A•A43U, U28C•A42G, C29A•G41U; the stem is identical to the Ser-AGA-19 stem in lane 1. (C) C34G substitution activates ASL representing tRNATrpCCA as a Mod5p substrate. Mod5p modification of Ser-AGA-19 (lane 1) or the scTrp-19 ASLs with the anticodon sequences indicated above the lanes (upper panel). (Lower panel) The ASLs gel in upper panel after staining with EtBr. (D) Parallel in vitro modification of the same set of ASLs by Mod5p (lanes 1–5) and Tit1p (lanes 6–10). (Lanes 1,6) Ser-AGA-19; (lanes 2,7) scTrp-ICA; (lanes 3,8) scTrp-CAA; (lanes 4,9) scTrp-CUA; (lanes 5,10) scTrp-UCA. (E–I) C34G substitution activates tRNATrpCCA as a Mod5p substrate in vivo. MOD5 and mod5-Δ cells were transformed with empty vector (lanes 1–4) or vector expressing an S. pombe gene encoding tRNATrpCCA (lanes 5–8) or the point mutated tRNATrpGCA (lanes 9–12) as indicated above the lanes. RNA from the transformed cells was fractionated and blotted, and the membrane was sequentially hybridized, stripped, and rehybridized with probes indicated to the right of the panels according to the PHA6 Northern blot assay.

Tek N. Lamichhane, et al. RNA. 2011 October;17(10):1846-1857.

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