Limits...
Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.

Duc C, Sherstnev A, Cole C, Barton GJ, Simpson GG - PLoS Genet. (2013)

Bottom Line: We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs.Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins.We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.

ABSTRACT
Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3' formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation.

Show MeSH
PIF5–PA03, an example of chimeric RNA formation controlled by FPA.(A) Normalised reads mapping to the locus encoding PIF5–PA03. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser [55] and corresponds to combined reads from the three sequenced biological replicates for each genotype. (B) Location of RNA gel blot probes are indicated by numbers (P1–P6) and the tested fusion region by a dotted line. (C) RT-PCR analysis of a contiguous RNA between PIF5 and PA03 detected in fpa-8. UBIQUITIN LIGASE 21 (UBC) was used as a control. (D,E) RNA gel blot analysis of PIF5–PA03 chimeric RNAs in wild-type (WT) and fpa-8. P, PIF5; C, PA03 transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Probes used are shown in (B). (F) 5′RACE analysis of the γ and γ′ RNAs with or without tobacco acid pyrophosphatase (TAP) treatment. PCR products were separated on an agarose gel and stained with ethidium bromide. (G) Schematic representation of the different RNAs expressed at the PIF5 locus in fpa mutants. The splicing event occurring in the β chimeric RNA is shown by a red line.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814327&req=5

pgen-1003867-g005: PIF5–PA03, an example of chimeric RNA formation controlled by FPA.(A) Normalised reads mapping to the locus encoding PIF5–PA03. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser [55] and corresponds to combined reads from the three sequenced biological replicates for each genotype. (B) Location of RNA gel blot probes are indicated by numbers (P1–P6) and the tested fusion region by a dotted line. (C) RT-PCR analysis of a contiguous RNA between PIF5 and PA03 detected in fpa-8. UBIQUITIN LIGASE 21 (UBC) was used as a control. (D,E) RNA gel blot analysis of PIF5–PA03 chimeric RNAs in wild-type (WT) and fpa-8. P, PIF5; C, PA03 transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Probes used are shown in (B). (F) 5′RACE analysis of the γ and γ′ RNAs with or without tobacco acid pyrophosphatase (TAP) treatment. PCR products were separated on an agarose gel and stained with ethidium bromide. (G) Schematic representation of the different RNAs expressed at the PIF5 locus in fpa mutants. The splicing event occurring in the β chimeric RNA is shown by a red line.

Mentions: Among those RNAs affected by FPA, we identified cases in which tandem genes showed reciprocal changes in read abundance. For example, in the fpa-7 mutant we found a reduction in the number of reads aligning to the 3′ end of At3g59060 (PHYTOCHROME INTERACTING FACTOR 5, PIF5) compared to WT, but the number of reads mapping to the 3′ end of the downstream gene At3g59050 (POLYAMINE OXIDASE 3, PA03) was increased (Figure 5A). We asked whether these read differences could be explained by defective 3′ end formation at PIF5 in fpa mutants, leading to chimeric RNAs being cleaved and polyadenylated within the 3′UTR of PA03. Consistent with this idea, RT-PCR analysis using primers anchored in PIF5 and PA03 (and thus spanning the intergenic region) detected the formation of chimeric RNAs specifically in fpa mutants (Figure 5B–C). RNA gel blot analysis confirmed this, with a probe to the 5′ end of PIF5 revealing 60% read-through into RNAs of increased size relative to PIF5 (Figure 5D). Three major hybridising signals specific to fpa mutants were detected using probes spanning the intergenic region, the PA03 3′UTR and the different exons and UTRs of PIF5 (Figure 5D–E). A combination of RT-PCR, 5′ rapid amplification of cDNA ends (RACE), RNA gel blot and cloning approaches identified two of these (α and β) as chimeric RNAs that differ as a result of a cryptic splicing event (Figure 5E–G), while the third comprised two similar sized RNAs with 5′ ends mapping to either the 3′UTR of PIF5 (γ) or the intergenic sequence (γ′) (Figure 5E–G). The differential sensitivities of these RNAs to tobacco acid pyrophosphatase (TAP) suggest that γ is capped, but γ′ is not (Figure 5F). None of the chimeric RNAs altered the deduced PIF5 open reading frame, but they did effectively extend the 3′UTR from 211 nt to 2,720 nt in α and to 1,680 nt in β chimeric transcripts. In contrast, native PA03 expression is undetectable in fpa mutants (Figure 5D). The 5′ end of γ RNAs aligned to the PIF5 3′UTR (Figure 5F,G) but, as judged by 5′RACE, was distinct from all of the 3′ ends that mapped to the PIF5 3′UTR (Figure 5G). This suggests that γ RNAs did not result from cleavage of chimeric RNAs followed by capping. Instead, we found differences between WT and fpa mutants in H3K4me3, a chromatin modification associated with transcription start sites [34], across PIF5 and PA03, with a decrease in H3K4me3 at the 5′ end of PA03 and an additional H3K4me3 peak detected at the 3′ end of PIF5 in fpa-8 (Figure 6A). These data are consistent with a shift in the PA03 transcription start site accompanying the chimeric RNAs detected here in fpa mutants.


Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.

Duc C, Sherstnev A, Cole C, Barton GJ, Simpson GG - PLoS Genet. (2013)

PIF5–PA03, an example of chimeric RNA formation controlled by FPA.(A) Normalised reads mapping to the locus encoding PIF5–PA03. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser [55] and corresponds to combined reads from the three sequenced biological replicates for each genotype. (B) Location of RNA gel blot probes are indicated by numbers (P1–P6) and the tested fusion region by a dotted line. (C) RT-PCR analysis of a contiguous RNA between PIF5 and PA03 detected in fpa-8. UBIQUITIN LIGASE 21 (UBC) was used as a control. (D,E) RNA gel blot analysis of PIF5–PA03 chimeric RNAs in wild-type (WT) and fpa-8. P, PIF5; C, PA03 transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Probes used are shown in (B). (F) 5′RACE analysis of the γ and γ′ RNAs with or without tobacco acid pyrophosphatase (TAP) treatment. PCR products were separated on an agarose gel and stained with ethidium bromide. (G) Schematic representation of the different RNAs expressed at the PIF5 locus in fpa mutants. The splicing event occurring in the β chimeric RNA is shown by a red line.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814327&req=5

pgen-1003867-g005: PIF5–PA03, an example of chimeric RNA formation controlled by FPA.(A) Normalised reads mapping to the locus encoding PIF5–PA03. Exons are denoted by coloured rectangles, UTRs by adjoining narrower rectangles and introns by lines. The image of normalised read alignments was made using the Integrated Genome Browser [55] and corresponds to combined reads from the three sequenced biological replicates for each genotype. (B) Location of RNA gel blot probes are indicated by numbers (P1–P6) and the tested fusion region by a dotted line. (C) RT-PCR analysis of a contiguous RNA between PIF5 and PA03 detected in fpa-8. UBIQUITIN LIGASE 21 (UBC) was used as a control. (D,E) RNA gel blot analysis of PIF5–PA03 chimeric RNAs in wild-type (WT) and fpa-8. P, PIF5; C, PA03 transcripts. β-TUBULIN (β-TUB.) was used as an internal control. Probes used are shown in (B). (F) 5′RACE analysis of the γ and γ′ RNAs with or without tobacco acid pyrophosphatase (TAP) treatment. PCR products were separated on an agarose gel and stained with ethidium bromide. (G) Schematic representation of the different RNAs expressed at the PIF5 locus in fpa mutants. The splicing event occurring in the β chimeric RNA is shown by a red line.
Mentions: Among those RNAs affected by FPA, we identified cases in which tandem genes showed reciprocal changes in read abundance. For example, in the fpa-7 mutant we found a reduction in the number of reads aligning to the 3′ end of At3g59060 (PHYTOCHROME INTERACTING FACTOR 5, PIF5) compared to WT, but the number of reads mapping to the 3′ end of the downstream gene At3g59050 (POLYAMINE OXIDASE 3, PA03) was increased (Figure 5A). We asked whether these read differences could be explained by defective 3′ end formation at PIF5 in fpa mutants, leading to chimeric RNAs being cleaved and polyadenylated within the 3′UTR of PA03. Consistent with this idea, RT-PCR analysis using primers anchored in PIF5 and PA03 (and thus spanning the intergenic region) detected the formation of chimeric RNAs specifically in fpa mutants (Figure 5B–C). RNA gel blot analysis confirmed this, with a probe to the 5′ end of PIF5 revealing 60% read-through into RNAs of increased size relative to PIF5 (Figure 5D). Three major hybridising signals specific to fpa mutants were detected using probes spanning the intergenic region, the PA03 3′UTR and the different exons and UTRs of PIF5 (Figure 5D–E). A combination of RT-PCR, 5′ rapid amplification of cDNA ends (RACE), RNA gel blot and cloning approaches identified two of these (α and β) as chimeric RNAs that differ as a result of a cryptic splicing event (Figure 5E–G), while the third comprised two similar sized RNAs with 5′ ends mapping to either the 3′UTR of PIF5 (γ) or the intergenic sequence (γ′) (Figure 5E–G). The differential sensitivities of these RNAs to tobacco acid pyrophosphatase (TAP) suggest that γ is capped, but γ′ is not (Figure 5F). None of the chimeric RNAs altered the deduced PIF5 open reading frame, but they did effectively extend the 3′UTR from 211 nt to 2,720 nt in α and to 1,680 nt in β chimeric transcripts. In contrast, native PA03 expression is undetectable in fpa mutants (Figure 5D). The 5′ end of γ RNAs aligned to the PIF5 3′UTR (Figure 5F,G) but, as judged by 5′RACE, was distinct from all of the 3′ ends that mapped to the PIF5 3′UTR (Figure 5G). This suggests that γ RNAs did not result from cleavage of chimeric RNAs followed by capping. Instead, we found differences between WT and fpa mutants in H3K4me3, a chromatin modification associated with transcription start sites [34], across PIF5 and PA03, with a decrease in H3K4me3 at the 5′ end of PA03 and an additional H3K4me3 peak detected at the 3′ end of PIF5 in fpa-8 (Figure 6A). These data are consistent with a shift in the PA03 transcription start site accompanying the chimeric RNAs detected here in fpa mutants.

Bottom Line: We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs.Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins.We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom.

ABSTRACT
Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3' formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation.

Show MeSH