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Genome-Wide Analysis of PAPS1-Dependent Polyadenylation Identifies Novel Roles for Functionally Specialized Poly(A) Polymerases in Arabidopsis thaliana.

Kappel C, Trost G, Czesnick H, Ramming A, Kolbe B, Vi SL, Bispo C, Becker JD, de Moor C, Lenhard M - PLoS Genet. (2015)

Bottom Line: When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis.Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1.Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.

View Article: PubMed Central - PubMed

Affiliation: Universität Potsdam, Institut für Biochemie und Biologie, Potsdam-Golm, Germany.

ABSTRACT
The poly(A) tail at 3' ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A)-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A)-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A)-polymerase isoforms. Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.

No MeSH data available.


Role of PAPS1 in ribosome biogenesis.Quantification of total RNA (left), combined 18S and 28S rRNA (middle), and remaining non-ribosomal RNA (right) in pg per pollen grain from paps1-3 mutant and PAPS1 pLat52::DsRED wild-type pollen grains produced by paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants. Pollen grains were flow-sorted based on DsRED fluorescence and extracted total RNA was subjected to Bioanalyzer quantification. RNA amounts were related to the known number of pollen grains in the sorted fractions. Values represent five biological replicates; black dot is the median value, open blue circles show outliers as determined using the R/lattice boxplot.stats function.
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pgen.1005474.g003: Role of PAPS1 in ribosome biogenesis.Quantification of total RNA (left), combined 18S and 28S rRNA (middle), and remaining non-ribosomal RNA (right) in pg per pollen grain from paps1-3 mutant and PAPS1 pLat52::DsRED wild-type pollen grains produced by paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants. Pollen grains were flow-sorted based on DsRED fluorescence and extracted total RNA was subjected to Bioanalyzer quantification. RNA amounts were related to the known number of pollen grains in the sorted fractions. Values represent five biological replicates; black dot is the median value, open blue circles show outliers as determined using the R/lattice boxplot.stats function.

Mentions: To test whether the reduced poly(A)-tail lengths of transcripts encoding ribosomal proteins (see above) were functionally relevant, we sought to measure ribosome content in paps1 mutants. As ribosomal RNA (rRNA) is rapidly degraded when not incorporated into functional ribosomes [40,41], the amount of ribosomal RNA can be used as a proxy for ribosome content. Quantifying the proportion of ribosomal RNA in total RNA samples by a BioAnalyzer RNA chip indicated that there was no significant change in the relative amount of ribosomal RNA in paps1-1 mutants (S6A Fig). As the paps1-1 mutant is not a allele, we sought to determine whether the complete loss of PAPS1 function in the paps1-3 T-DNA insertion mutant would cause an effect on ribosome biogenesis [32]. As no paps1-3 homozygous mutants can be obtained due to a male gametophytic defect, we used a fluorescence-marking system [42] to sort paps1-3 mutant and wild-type pollen from heterozygous plants using fluorescence-activated cell sorting (FACS) [43]. A line carrying a pollen-expressed pLat52::DsRED reporter construct inserted about 290 kb from the wild-type PAPS1 allele was crossed to paps1-3 heterozygous plants, and paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants were selected. After meiosis, these plants produce roughly 50% paps1-3 mutant pollen lacking DsRED fluorescence and roughly 50% PAPS1 wild-type pollen with DsRED fluorescence (S6B Fig), as well as a negligible number of recombinant pollen grains. Isolation of total RNA from FACS-sorted pollen grains and Bioanalyzer analysis indicated that paps1-3 mutant pollen grains contain a significantly reduced total RNA amount per pollen grain, which is due to a specific reduction in ribosomal RNAs, while non-rRNA was very similar in amount per pollen grain between the two genotypes (Fig 3). Thus, characterization of this -mutant defect suggests that the shortened tails of transcripts encoding ribosomal proteins cause a reduced accumulation of these proteins and, as a consequence, of ribosomal RNAs in mature ribosomes.


Genome-Wide Analysis of PAPS1-Dependent Polyadenylation Identifies Novel Roles for Functionally Specialized Poly(A) Polymerases in Arabidopsis thaliana.

Kappel C, Trost G, Czesnick H, Ramming A, Kolbe B, Vi SL, Bispo C, Becker JD, de Moor C, Lenhard M - PLoS Genet. (2015)

Role of PAPS1 in ribosome biogenesis.Quantification of total RNA (left), combined 18S and 28S rRNA (middle), and remaining non-ribosomal RNA (right) in pg per pollen grain from paps1-3 mutant and PAPS1 pLat52::DsRED wild-type pollen grains produced by paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants. Pollen grains were flow-sorted based on DsRED fluorescence and extracted total RNA was subjected to Bioanalyzer quantification. RNA amounts were related to the known number of pollen grains in the sorted fractions. Values represent five biological replicates; black dot is the median value, open blue circles show outliers as determined using the R/lattice boxplot.stats function.
© Copyright Policy
Related In: Results  -  Collection

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pgen.1005474.g003: Role of PAPS1 in ribosome biogenesis.Quantification of total RNA (left), combined 18S and 28S rRNA (middle), and remaining non-ribosomal RNA (right) in pg per pollen grain from paps1-3 mutant and PAPS1 pLat52::DsRED wild-type pollen grains produced by paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants. Pollen grains were flow-sorted based on DsRED fluorescence and extracted total RNA was subjected to Bioanalyzer quantification. RNA amounts were related to the known number of pollen grains in the sorted fractions. Values represent five biological replicates; black dot is the median value, open blue circles show outliers as determined using the R/lattice boxplot.stats function.
Mentions: To test whether the reduced poly(A)-tail lengths of transcripts encoding ribosomal proteins (see above) were functionally relevant, we sought to measure ribosome content in paps1 mutants. As ribosomal RNA (rRNA) is rapidly degraded when not incorporated into functional ribosomes [40,41], the amount of ribosomal RNA can be used as a proxy for ribosome content. Quantifying the proportion of ribosomal RNA in total RNA samples by a BioAnalyzer RNA chip indicated that there was no significant change in the relative amount of ribosomal RNA in paps1-1 mutants (S6A Fig). As the paps1-1 mutant is not a allele, we sought to determine whether the complete loss of PAPS1 function in the paps1-3 T-DNA insertion mutant would cause an effect on ribosome biogenesis [32]. As no paps1-3 homozygous mutants can be obtained due to a male gametophytic defect, we used a fluorescence-marking system [42] to sort paps1-3 mutant and wild-type pollen from heterozygous plants using fluorescence-activated cell sorting (FACS) [43]. A line carrying a pollen-expressed pLat52::DsRED reporter construct inserted about 290 kb from the wild-type PAPS1 allele was crossed to paps1-3 heterozygous plants, and paps1-3—/ PAPS1 pLat52::DsRED transheterozygous plants were selected. After meiosis, these plants produce roughly 50% paps1-3 mutant pollen lacking DsRED fluorescence and roughly 50% PAPS1 wild-type pollen with DsRED fluorescence (S6B Fig), as well as a negligible number of recombinant pollen grains. Isolation of total RNA from FACS-sorted pollen grains and Bioanalyzer analysis indicated that paps1-3 mutant pollen grains contain a significantly reduced total RNA amount per pollen grain, which is due to a specific reduction in ribosomal RNAs, while non-rRNA was very similar in amount per pollen grain between the two genotypes (Fig 3). Thus, characterization of this -mutant defect suggests that the shortened tails of transcripts encoding ribosomal proteins cause a reduced accumulation of these proteins and, as a consequence, of ribosomal RNAs in mature ribosomes.

Bottom Line: When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis.Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1.Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.

View Article: PubMed Central - PubMed

Affiliation: Universität Potsdam, Institut für Biochemie und Biologie, Potsdam-Golm, Germany.

ABSTRACT
The poly(A) tail at 3' ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A)-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A)-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A)-polymerase isoforms. Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.

No MeSH data available.