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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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The RES complex promotes splicing of TAN1 pre-mRNA. (A) Northern analysis of total RNA isolated from wild-type (UMY2219), bud13Δ (MJY546), snu17Δ (MJY548) and pml1Δ (MJY535) cells grown in YEPD medium at 30°C. The blot was probed for TAN1 and PGK1 transcripts using randomly labeled DNA fragments. 18S rRNA was detected using an oligonucleotide probe. (B) Western analysis of the indicated strains (MJY664, MJY666, MJY668 and MJY670) grown in YEPD medium at 30°C. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins. Control experiments showed that the sequence for the 3HA-tag did not alter the efficiency of which the TAN1 pre-mRNA is spliced in either wild-type or RES-deficient cells (data not shown). (C) HPLC analysis of nucleosides of total tRNA isolated from bud13Δ tan1Δ (MJY568) cells carrying an empty low-copy (l.c.) URA3 vector or the same plasmid containing the BUD13, TAN1 or TAN1Δi gene. Cells were grown in SC-ura medium at 30°C.
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gkt269-F3: The RES complex promotes splicing of TAN1 pre-mRNA. (A) Northern analysis of total RNA isolated from wild-type (UMY2219), bud13Δ (MJY546), snu17Δ (MJY548) and pml1Δ (MJY535) cells grown in YEPD medium at 30°C. The blot was probed for TAN1 and PGK1 transcripts using randomly labeled DNA fragments. 18S rRNA was detected using an oligonucleotide probe. (B) Western analysis of the indicated strains (MJY664, MJY666, MJY668 and MJY670) grown in YEPD medium at 30°C. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins. Control experiments showed that the sequence for the 3HA-tag did not alter the efficiency of which the TAN1 pre-mRNA is spliced in either wild-type or RES-deficient cells (data not shown). (C) HPLC analysis of nucleosides of total tRNA isolated from bud13Δ tan1Δ (MJY568) cells carrying an empty low-copy (l.c.) URA3 vector or the same plasmid containing the BUD13, TAN1 or TAN1Δi gene. Cells were grown in SC-ura medium at 30°C.

Mentions: As there is no evidence to suggest a direct role of the RES complex in tRNA modification, it seemed possible that the ac4C-deficiency in bud13Δ, snu17Δ and pml1Δ mutants might be a consequence of defects in pre-mRNA splicing and/or nuclear retention. In fact, the tRNA modification defect of the individual mutants correlates to the importance of respective factor in splicing, i.e. the lack of Bud13p or Snu17p generates stronger splicing defects than a lack of Pml1p (4). Interestingly, the TAN1 gene, which is the only, to date, identified gene required for formation of ac4C in tRNA (27), harbors a 58 nt intron at the 5′ part of the ORF. To investigate if the RES complex controls ac4C levels in tRNA by promoting TAN1 pre-mRNA splicing, we used northern blotting to analyze TAN1 transcripts in wild-type, bud13Δ, snu17Δ and pml1Δ cells. The blots were also probed for the intron-less PGK1 mRNA and 18S rRNA, which served as a loading control. The analyses revealed an accumulation of unspliced TAN1 pre-mRNA in all three mutants of which the bud13Δ and snu17Δ strains showed spliced TAN1 mRNA levels below the level of detection (Figure 3A). The TAN1 pre-mRNA splicing defect in pml1Δ cells was enhanced at 37°C (data not shown), explaining the reduced abundance of ac4C at the elevated temperature. Western blot analyses of strains in which the DNA sequence for three tandem influenza virus hemagglutinin epitopes (3HA) was fused to the endogenous TAN1 ORF showed that the Tan1 protein levels were reduced in pml1Δ and not detectable in bud13Δ or snu17Δ cells (Figure 3B). Collectively, these results support a model in which the ac4C-deficiency in RES mutants is caused by inefficient splicing of TAN1 pre-mRNA.Figure 3.


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

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

The RES complex promotes splicing of TAN1 pre-mRNA. (A) Northern analysis of total RNA isolated from wild-type (UMY2219), bud13Δ (MJY546), snu17Δ (MJY548) and pml1Δ (MJY535) cells grown in YEPD medium at 30°C. The blot was probed for TAN1 and PGK1 transcripts using randomly labeled DNA fragments. 18S rRNA was detected using an oligonucleotide probe. (B) Western analysis of the indicated strains (MJY664, MJY666, MJY668 and MJY670) grown in YEPD medium at 30°C. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins. Control experiments showed that the sequence for the 3HA-tag did not alter the efficiency of which the TAN1 pre-mRNA is spliced in either wild-type or RES-deficient cells (data not shown). (C) HPLC analysis of nucleosides of total tRNA isolated from bud13Δ tan1Δ (MJY568) cells carrying an empty low-copy (l.c.) URA3 vector or the same plasmid containing the BUD13, TAN1 or TAN1Δi gene. Cells were grown in SC-ura medium at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675484&req=5

gkt269-F3: The RES complex promotes splicing of TAN1 pre-mRNA. (A) Northern analysis of total RNA isolated from wild-type (UMY2219), bud13Δ (MJY546), snu17Δ (MJY548) and pml1Δ (MJY535) cells grown in YEPD medium at 30°C. The blot was probed for TAN1 and PGK1 transcripts using randomly labeled DNA fragments. 18S rRNA was detected using an oligonucleotide probe. (B) Western analysis of the indicated strains (MJY664, MJY666, MJY668 and MJY670) grown in YEPD medium at 30°C. Monoclonal antibodies against HA or Pgk1p were used to detect the indicated proteins. Control experiments showed that the sequence for the 3HA-tag did not alter the efficiency of which the TAN1 pre-mRNA is spliced in either wild-type or RES-deficient cells (data not shown). (C) HPLC analysis of nucleosides of total tRNA isolated from bud13Δ tan1Δ (MJY568) cells carrying an empty low-copy (l.c.) URA3 vector or the same plasmid containing the BUD13, TAN1 or TAN1Δi gene. Cells were grown in SC-ura medium at 30°C.
Mentions: As there is no evidence to suggest a direct role of the RES complex in tRNA modification, it seemed possible that the ac4C-deficiency in bud13Δ, snu17Δ and pml1Δ mutants might be a consequence of defects in pre-mRNA splicing and/or nuclear retention. In fact, the tRNA modification defect of the individual mutants correlates to the importance of respective factor in splicing, i.e. the lack of Bud13p or Snu17p generates stronger splicing defects than a lack of Pml1p (4). Interestingly, the TAN1 gene, which is the only, to date, identified gene required for formation of ac4C in tRNA (27), harbors a 58 nt intron at the 5′ part of the ORF. To investigate if the RES complex controls ac4C levels in tRNA by promoting TAN1 pre-mRNA splicing, we used northern blotting to analyze TAN1 transcripts in wild-type, bud13Δ, snu17Δ and pml1Δ cells. The blots were also probed for the intron-less PGK1 mRNA and 18S rRNA, which served as a loading control. The analyses revealed an accumulation of unspliced TAN1 pre-mRNA in all three mutants of which the bud13Δ and snu17Δ strains showed spliced TAN1 mRNA levels below the level of detection (Figure 3A). The TAN1 pre-mRNA splicing defect in pml1Δ cells was enhanced at 37°C (data not shown), explaining the reduced abundance of ac4C at the elevated temperature. Western blot analyses of strains in which the DNA sequence for three tandem influenza virus hemagglutinin epitopes (3HA) was fused to the endogenous TAN1 ORF showed that the Tan1 protein levels were reduced in pml1Δ and not detectable in bud13Δ or snu17Δ cells (Figure 3B). Collectively, these results support a model in which the ac4C-deficiency in RES mutants is caused by inefficient splicing of TAN1 pre-mRNA.Figure 3.

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