Detection of polyadenylated tRNA_i^Met in trm6-504 and trm6-504 suppressor mutants. Total RNA and poly(A)⁺ RNA were isolated from wild-type (Y200), trm6-504(Y190), trm6-504 trf4-20 (sup1), and trm6-504 rrp44-20(sup2) strains grown in YPD at 30°C as detailed in Materials and Methods. Total RNA (5 μg) and poly(A)⁺ RNA (2–4 μg) were separated on a 6% polyacrylamide (19:1) 8 Murea gel. Membrane-bound tRNA_i^Met was detected using a radiolabeled oligonucleotide JA11 followed by autoradiography.

Internally truncated U6 snRNA is a substrate for Trf4p polyadenylation and degradation by the nuclear exosome. Wild type (F39), rrp6Δ (F23), trf4Δ (F22), and trf4Δ + Sctrf4-236 were transformed with a single-copy number plasmid containing a mutant SNR6 gene (snr6Δ59–72). Total RNA (5 μg) from single transformants and a wildtype strain bearing an empty plasmid were analyzed by Northern hybridization using a radiolabeled probe (JA242) that recognizes the endogenous and plasmid-borne U6 snRNAs. The migration positions of the endogenous and snr6Δ59–72 mutant U6 snRNAs are shown for clarity.

Polyadenylated pre-tRNA_i^Met contains the 5′ leader sequence. (A) Northern hybridization analysis of total RNA (5 μg) and poly(A)⁺ RNA (2–4 μg) from trm6-504 (Y190) and wild type (Y200). (B) Northern hybridization of total RNA (5 μg) and poly(A)+ RNA (2–4 μg) from trm6-504 rrp6Δ (Y298) with (+) or without (−) pretreatment with oligo d(T) and RNaseH. The blots were hybridized with radiolabeled probes (JA382, JA72, JA66, and JA48) that recognize only pre-tRNA_i^Met containing the 5′ leader (A,B, lanes 1–3) or a radiolabeled probe (JA11) that is complementary to an internal portion of tRNA_i^Met (B, lanes 4–6). The point of migration of pre-tRNA_i^Met and mature tRNA_i^Met are shown for clarity.

Precursor form of hypomodified tRNA_i^Met is being polyadenylated. (A) An abbreviated and stylized representation of the tRNA_i^Met coding sequence with the predicted 3′ ends of each pre-tRNA_i^Met synthesized from IMT1–4 shown. The predicted transcription termination sites of pre-tRNA_i^Met are shown as 5–7 tandem Ts (Us in the transcript) are shown in bold. (B) The abbreviated coding sequence is followed by the exact sequence found at the 3′ end of each cDNA, and the poly(A) tail lengths are shown as a range (16–30), but the majority of the poly(A) tails were 16–19 As in length. The number of clones with the identical 3′ end sequence are shown in parentheses. (C) The abbreviated coding sequence for the truncated cDNAs found end with the last nucleotide of the tRNA (C⁶⁵ or C⁶⁰) followed by the poly(A) tail length for each.

Trf4p and Trf5p have overlapping functions in the degradation of hypomethylated tRNA_i^Met. (A) Yeast strains wild type (Y200), trm6-504 (Y190), trm6-504 trf4-20 (sup1), and trm6-504 trf5Δ were grown to saturation in YPD and serial 10-fold dilutions were plated onto YPD plates and incubated at 26°C or 36°C for 3 d and 2 d, respectively. (B) The same strains from A transformed with empty vector (WT, trm6-504, and trm6-504 trf5Δ) or a high-copy number plasmid containing TRF5 (WT, trm6-504, and trm6-504 trf5Δ + hcTRF5) were streaked to synthetic complete media lacking uracil and incubated at 30°C and 36°C for 3 d. (C) Northern hybridization of total RNA (3 μg) and poly(A)⁺ RNA (4–5 μg) isolated from wild-type (Y200), trm6-504 trf4Δ and trm6-504 trf4Δ + hcTRF5. The blot was hybridized with a radiolabeled oligonucleotide (JA11) to detect tRNA_i^Met.

Trf4p dependent polyadenylation of 3′ end truncated 5S rRNA. (A) A schematic diagram of 5S rDNA showing the coding region (filled box) and the 5′ and 3′ intergenic regions (lines). The approximate positions (not to scale) complementary to the oligonucleotide probes used in this study are shown as lines beneath the coding region. (B) Total RNA (5 μg) isolated from wild type (F39), rrp6Δ (F23), rrp44-20 (Y303), trf4Δ (F22), and trm6-504/trf4Δ (Y301) strains grown at 30°C was used for Northern hybridization analysis. The membrane was probed with JA99 to detect mature 5S rRNA, JA133 to detect 5S rRNA containing the first 20 nt of 5S rRNA and JA134 to detect 5S rRNA containing the last 20 nt. The 5S rRNA was visualized by autoradiography. (C) Total RNA (5 μg, lanes 1–5) and poly(A)+ RNA (2–4 μg, lanes 6–10) isolated from the same strains described in B was used for Northern hybridization analysis using probes JA133 (top) and JA134 (bottom). Indicated are the migration positions of the mature 5S rRNA, 3′-end truncated 5S rRNA, and poly(A)⁺ 5S rRNA, as well as the probe used for each hybridization.

The DXD motif of Trf4p is crucial for its poly(A) polymerase activity and the degradation of tRNA_i^Met. (A) Sctrf4-236 complements the cold sensitivity of trf4Δ. A strain bearing chromosomal trf4Δ was transformed with YCplac33, ScTRF4 (B254), or Sctrf4-236 (B316), and the transformants were grown on SC-URA at 30°C, then replica-printed onto SC-URA plates and incubated at 16° and 26°C for 3 d and 1 d, respectively. (B) trf4-236 suppresses the Ts⁻ phenotype of a trm6-504 mutant. A trm6-504 trf4Δ strain (Y301) was transformed with YCpLac33, ScTRF4 (B254), and Sctrf4-236 (B316); wild type (Y200) and trm6-504 (Y190) were transformed just with YCpLac33. Individual transformants were grown to saturation in SC-URA and 10-fold serial dilutions were spotted on SC-URA plates and incubated at 26°, 36°, and 16°C for 2, 3, and 4 d, respectively. (C) Northern hybridization of total RNA (5 μg) from wild type (Y200), trm6-504(Y190), trm6-504 trf4Δ (sup1) with YCpLac33, or Sctrf4-236 strains. Total RNA (5 μg) was separated on a 6% polyacrylamide (19:1) 8 Murea gel and transferred to a membrane and probed with JA11 to detect tRNA_i^Met and JA 151 to detect tRNA^Leu. The tRNAs were visualized by autoradiography and the quantities were determined by phosphorimage analysis using a STORM 640 and ImageQuant software (GE Healthcare). The quantity of tRNA_i^Met in each sample is shown as a percentage of tRNA_i^Met in the wild-type strain after normalization with tRNA_CAA^Leu. This experiment has been repeated and the quantity of tRNA_i^Met was found to be similar each time. (D) The DXD motif is important for its poly(A) polymerase activity in vivo. Total RNA (5 μg) and poly(A)⁺ RNA (2–4 μg) isolated from the transformants described in B were separated, and a Northern hybridization was conducted to detect tRNA_i^Met using a radiolabeled oligonucleotide (JA11) and autoradiography.