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The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression.

Zhou Y, Chen C, Johansson MJ - Nucleic Acids Res. (2013)

Bottom Line: The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine.We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C.Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.

ABSTRACT
The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA and that a lack of Pml1p reduces ac(4)C levels at elevated temperatures. The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

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Mutations in the BUD13 gene reduce the levels of ac4C in tRNA. (A) Schematic secondary structure of . The alteration caused by the sup61-T47:2C allele and the position of ac4C are indicated. (B) Growth of strains with sup61-T47:2C and/or bud13Δ alleles. The wild-type (UMY2219), sup61-T47:2C (UMY2256), bud13Δ (MJY546) and sup61-T47:2C bud13Δ (MJY553) strains were grown overnight in YEPD medium, serially diluted, spotted on YEPD plates and incubated at 25°C, 30°C or 37°C for 2 days. (C) Northern analysis of total RNA isolated from the strains described in B grown in YEPD medium at 25°C. The blot was probed for pre-,  and  using oligonucleotide probes.  serves as a loading control. (D) HPLC analyses of tRNA-derived nucleosides from wild-type (UMY2219), tan1Δ (MJY550) and bud13Δ (MJY546) cells carrying the indicated low-copy (l.c.) URA3 plasmids. Cells were grown at 30°C in synthetic complete medium lacking uracil (SC-ura).
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gkt269-F1: Mutations in the BUD13 gene reduce the levels of ac4C in tRNA. (A) Schematic secondary structure of . The alteration caused by the sup61-T47:2C allele and the position of ac4C are indicated. (B) Growth of strains with sup61-T47:2C and/or bud13Δ alleles. The wild-type (UMY2219), sup61-T47:2C (UMY2256), bud13Δ (MJY546) and sup61-T47:2C bud13Δ (MJY553) strains were grown overnight in YEPD medium, serially diluted, spotted on YEPD plates and incubated at 25°C, 30°C or 37°C for 2 days. (C) Northern analysis of total RNA isolated from the strains described in B grown in YEPD medium at 25°C. The blot was probed for pre-, and using oligonucleotide probes. serves as a loading control. (D) HPLC analyses of tRNA-derived nucleosides from wild-type (UMY2219), tan1Δ (MJY550) and bud13Δ (MJY546) cells carrying the indicated low-copy (l.c.) URA3 plasmids. Cells were grown at 30°C in synthetic complete medium lacking uracil (SC-ura).

Mentions: To identify factors promoting maturation of tRNA, we previously performed a screen for genes required for growth of cells harboring a sup61-T47:2C allele, which codes for a species with an alteration in the variable arm (Figure 1A) (27). The screen identified mutants representing 12 different complementation groups of which three groups consisted of strains with mutations in genes for already characterized tRNA modifying enzymes (DUS2, MOD5 and TRM1) and one defined strains with mutations in the TAN1 gene, which we subsequently showed to be required for formation of ac4C12 in tRNA (Figure 1A) (23,27). By using a yeast genomic library, we found that a mutant in one of the remaining eight groups was complemented by the BUD13 gene, which codes for a subunit of the RES complex. To demonstrate unambiguously that Bud13p is required for growth of cells with the altered form of , we combined the sup61-T47:2C mutation with a bud13Δ allele. The resulting sup61-T47:2C bud13Δ double mutants were slow-growing at 25°C and inviable at 30°C and 37°C (Figure 1B). To investigate the mechanism by which Bud13p influences viability of sup61-T47:2C cells, we used northern blotting to determine the levels of in wild-type, sup61-T47:2C, bud13Δ and bud13Δ sup61-T47:2C cells. The analyses revealed that the combination of bud13Δ and sup61-T47:2C alleles caused a synergistic reduction in levels (Figure 1C), indicating that Bud13p may be important for tRNA maturation and/or stability.Figure 1.


The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression.

Zhou Y, Chen C, Johansson MJ - Nucleic Acids Res. (2013)

Mutations in the BUD13 gene reduce the levels of ac4C in tRNA. (A) Schematic secondary structure of . The alteration caused by the sup61-T47:2C allele and the position of ac4C are indicated. (B) Growth of strains with sup61-T47:2C and/or bud13Δ alleles. The wild-type (UMY2219), sup61-T47:2C (UMY2256), bud13Δ (MJY546) and sup61-T47:2C bud13Δ (MJY553) strains were grown overnight in YEPD medium, serially diluted, spotted on YEPD plates and incubated at 25°C, 30°C or 37°C for 2 days. (C) Northern analysis of total RNA isolated from the strains described in B grown in YEPD medium at 25°C. The blot was probed for pre-,  and  using oligonucleotide probes.  serves as a loading control. (D) HPLC analyses of tRNA-derived nucleosides from wild-type (UMY2219), tan1Δ (MJY550) and bud13Δ (MJY546) cells carrying the indicated low-copy (l.c.) URA3 plasmids. Cells were grown at 30°C in synthetic complete medium lacking uracil (SC-ura).
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Related In: Results  -  Collection

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gkt269-F1: Mutations in the BUD13 gene reduce the levels of ac4C in tRNA. (A) Schematic secondary structure of . The alteration caused by the sup61-T47:2C allele and the position of ac4C are indicated. (B) Growth of strains with sup61-T47:2C and/or bud13Δ alleles. The wild-type (UMY2219), sup61-T47:2C (UMY2256), bud13Δ (MJY546) and sup61-T47:2C bud13Δ (MJY553) strains were grown overnight in YEPD medium, serially diluted, spotted on YEPD plates and incubated at 25°C, 30°C or 37°C for 2 days. (C) Northern analysis of total RNA isolated from the strains described in B grown in YEPD medium at 25°C. The blot was probed for pre-, and using oligonucleotide probes. serves as a loading control. (D) HPLC analyses of tRNA-derived nucleosides from wild-type (UMY2219), tan1Δ (MJY550) and bud13Δ (MJY546) cells carrying the indicated low-copy (l.c.) URA3 plasmids. Cells were grown at 30°C in synthetic complete medium lacking uracil (SC-ura).
Mentions: To identify factors promoting maturation of tRNA, we previously performed a screen for genes required for growth of cells harboring a sup61-T47:2C allele, which codes for a species with an alteration in the variable arm (Figure 1A) (27). The screen identified mutants representing 12 different complementation groups of which three groups consisted of strains with mutations in genes for already characterized tRNA modifying enzymes (DUS2, MOD5 and TRM1) and one defined strains with mutations in the TAN1 gene, which we subsequently showed to be required for formation of ac4C12 in tRNA (Figure 1A) (23,27). By using a yeast genomic library, we found that a mutant in one of the remaining eight groups was complemented by the BUD13 gene, which codes for a subunit of the RES complex. To demonstrate unambiguously that Bud13p is required for growth of cells with the altered form of , we combined the sup61-T47:2C mutation with a bud13Δ allele. The resulting sup61-T47:2C bud13Δ double mutants were slow-growing at 25°C and inviable at 30°C and 37°C (Figure 1B). To investigate the mechanism by which Bud13p influences viability of sup61-T47:2C cells, we used northern blotting to determine the levels of in wild-type, sup61-T47:2C, bud13Δ and bud13Δ sup61-T47:2C cells. The analyses revealed that the combination of bud13Δ and sup61-T47:2C alleles caused a synergistic reduction in levels (Figure 1C), indicating that Bud13p may be important for tRNA maturation and/or stability.Figure 1.

Bottom Line: The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine.We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C.Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.

ABSTRACT
The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA and that a lack of Pml1p reduces ac(4)C levels at elevated temperatures. The ac(4)C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac(4)C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac(4)C in tRNA.

Show MeSH
Related in: MedlinePlus