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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.

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FPA affects intronic cleavage site selection and intergenic read-through.(A) Reads mapping to the locus encoding FPA. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated, as are the poly(A) sites ‘T’ resulting from the T-DNA insertion in fpa-7. (B, C) Nucleotide composition profiles around cleavage sites within annotated genes (B) and at intergenic sites (C) display alternating A- and U-rich sequences. USE, upstream sequence element; PAS, poly(A) signal; Fip1, the U-rich sequence upstream of the cleavage site is the proposed binding site of FIP1 [58]; DSE, downstream sequence element. (D–F) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (D). The upper panel shows reads mapping to the (+) strand 3′ end of At1g29520, while the lower panel shows reads mapping to the (−) strand 3′ end of At1g29530. (E) R1 and R2 contiguous RNAs were validated by RT-PCR (red dashed lines) with poly(A)+ RNA. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. (F) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red narrower rectangles represent regions specific to the read-through transcript and red lines the 3′UTR introns. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.
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pgen-1003867-g004: FPA affects intronic cleavage site selection and intergenic read-through.(A) Reads mapping to the locus encoding FPA. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated, as are the poly(A) sites ‘T’ resulting from the T-DNA insertion in fpa-7. (B, C) Nucleotide composition profiles around cleavage sites within annotated genes (B) and at intergenic sites (C) display alternating A- and U-rich sequences. USE, upstream sequence element; PAS, poly(A) signal; Fip1, the U-rich sequence upstream of the cleavage site is the proposed binding site of FIP1 [58]; DSE, downstream sequence element. (D–F) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (D). The upper panel shows reads mapping to the (+) strand 3′ end of At1g29520, while the lower panel shows reads mapping to the (−) strand 3′ end of At1g29530. (E) R1 and R2 contiguous RNAs were validated by RT-PCR (red dashed lines) with poly(A)+ RNA. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. (F) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red narrower rectangles represent regions specific to the read-through transcript and red lines the 3′UTR introns. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.

Mentions: Besides refining our understanding of previously proposed roles for FPA in flowering and RNA silencing, we sought to identify the generic consequences of disrupting a regulator of RNA 3′ end formation. For example, one might predict that 3′ end formation within intronic sites and conventional 3′UTRs would be altered in fpa mutants. Cleavage and polyadenylation within intronic sequences outside the 3′UTR can have profound consequences on gene function by truncating mRNA coding potential. FPA effects autoregulation in this way by mediating selection of the promoter proximal intronic cleavage site within its own pre-mRNA [14]. We therefore asked whether FPA controls alternative polyadenylation at other intronic sites. We applied our data-smoothing and peak-finding algorithms to define cleavage sites [22] and estimated differential usage of these sites using DESeq. The validity of this approach was supported by the finding that previously identified intronic alternative polyadenylation events within FPA (Figure 4A; P = 7.10e−11), but not FCA were dependent on FPA function [14], [22]. Unexpectedly, additional intronic cleavage sites were also detected in FPA RNA itself, but only in the transfer (T)-DNA-induced fpa-7 allele (Figure 4A) and not in ethyl methanesulfonate (EMS)-induced alleles such as fpa-8[21], suggesting that T-DNA insertions can trigger cryptic cleavage and polyadenylation (Figure S3A–F). These allele-dependent distinctions in patterns of FPA polyadenylation were indicated by previous RNA gel blot hybridisations [14]. Reduced selection of intronic cleavage sites in 13 genes and increased selection of intronic cleavage sites in another 25 genes were found in fpa-7 (Table S4), indicating that FPA ultimately promotes cleavage at some sites, but represses it at others.


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

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

FPA affects intronic cleavage site selection and intergenic read-through.(A) Reads mapping to the locus encoding FPA. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated, as are the poly(A) sites ‘T’ resulting from the T-DNA insertion in fpa-7. (B, C) Nucleotide composition profiles around cleavage sites within annotated genes (B) and at intergenic sites (C) display alternating A- and U-rich sequences. USE, upstream sequence element; PAS, poly(A) signal; Fip1, the U-rich sequence upstream of the cleavage site is the proposed binding site of FIP1 [58]; DSE, downstream sequence element. (D–F) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (D). The upper panel shows reads mapping to the (+) strand 3′ end of At1g29520, while the lower panel shows reads mapping to the (−) strand 3′ end of At1g29530. (E) R1 and R2 contiguous RNAs were validated by RT-PCR (red dashed lines) with poly(A)+ RNA. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. (F) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red narrower rectangles represent regions specific to the read-through transcript and red lines the 3′UTR introns. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003867-g004: FPA affects intronic cleavage site selection and intergenic read-through.(A) Reads mapping to the locus encoding FPA. Promoter proximal ‘P’ and distal ‘D’ alternative poly(A) sites are indicated, as are the poly(A) sites ‘T’ resulting from the T-DNA insertion in fpa-7. (B, C) Nucleotide composition profiles around cleavage sites within annotated genes (B) and at intergenic sites (C) display alternating A- and U-rich sequences. USE, upstream sequence element; PAS, poly(A) signal; Fip1, the U-rich sequence upstream of the cleavage site is the proposed binding site of FIP1 [58]; DSE, downstream sequence element. (D–F) Example of intergenic DRS reads mapping antisense to a coding gene. Normalised reads mapping to the At1g29520–At1g29530 loci are displayed in (D). The upper panel shows reads mapping to the (+) strand 3′ end of At1g29520, while the lower panel shows reads mapping to the (−) strand 3′ end of At1g29530. (E) R1 and R2 contiguous RNAs were validated by RT-PCR (red dashed lines) with poly(A)+ RNA. RT-PCR products were separated on agarose gels and stained with ethidium bromide. Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. (F) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red narrower rectangles represent regions specific to the read-through transcript and red lines the 3′UTR introns. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.
Mentions: Besides refining our understanding of previously proposed roles for FPA in flowering and RNA silencing, we sought to identify the generic consequences of disrupting a regulator of RNA 3′ end formation. For example, one might predict that 3′ end formation within intronic sites and conventional 3′UTRs would be altered in fpa mutants. Cleavage and polyadenylation within intronic sequences outside the 3′UTR can have profound consequences on gene function by truncating mRNA coding potential. FPA effects autoregulation in this way by mediating selection of the promoter proximal intronic cleavage site within its own pre-mRNA [14]. We therefore asked whether FPA controls alternative polyadenylation at other intronic sites. We applied our data-smoothing and peak-finding algorithms to define cleavage sites [22] and estimated differential usage of these sites using DESeq. The validity of this approach was supported by the finding that previously identified intronic alternative polyadenylation events within FPA (Figure 4A; P = 7.10e−11), but not FCA were dependent on FPA function [14], [22]. Unexpectedly, additional intronic cleavage sites were also detected in FPA RNA itself, but only in the transfer (T)-DNA-induced fpa-7 allele (Figure 4A) and not in ethyl methanesulfonate (EMS)-induced alleles such as fpa-8[21], suggesting that T-DNA insertions can trigger cryptic cleavage and polyadenylation (Figure S3A–F). These allele-dependent distinctions in patterns of FPA polyadenylation were indicated by previous RNA gel blot hybridisations [14]. Reduced selection of intronic cleavage sites in 13 genes and increased selection of intronic cleavage sites in another 25 genes were found in fpa-7 (Table S4), indicating that FPA ultimately promotes cleavage at some sites, but represses it at others.

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