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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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Distribution of DRS reads.(A) Genome-wide distribution after re-annotation in wild-type (WT) and fpa-7. (B) Distribution of DRS reads mapping to protein-coding genes after re-annotation.
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pgen-1003867-g001: Distribution of DRS reads.(A) Genome-wide distribution after re-annotation in wild-type (WT) and fpa-7. (B) Distribution of DRS reads mapping to protein-coding genes after re-annotation.

Mentions: We subjected total RNA purified from three biological replicates of WT A. thaliana [Columbia-0 (Col-0) accession] and fpa-7 loss-of-function mutants to DRS. RNA was prepared from 14-day-old whole seedlings. A total of 22,560,508 WT and 24,383,585 fpa-7 reads that map polyadenylated RNA 3′ ends were aligned uniquely to the most recent A. thaliana genome release (currently TAIR10). A summary of the read statistics is given in Table S1. In each genotype, the vast majority of reads mapped to 3′ untranslated regions (UTRs) of protein-coding genes (Figure 1A–B). The DRS data can be visualised using aligned reads available at www.compbio.dundee.ac.uk/polyADB/.


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

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

Distribution of DRS reads.(A) Genome-wide distribution after re-annotation in wild-type (WT) and fpa-7. (B) Distribution of DRS reads mapping to protein-coding genes after re-annotation.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003867-g001: Distribution of DRS reads.(A) Genome-wide distribution after re-annotation in wild-type (WT) and fpa-7. (B) Distribution of DRS reads mapping to protein-coding genes after re-annotation.
Mentions: We subjected total RNA purified from three biological replicates of WT A. thaliana [Columbia-0 (Col-0) accession] and fpa-7 loss-of-function mutants to DRS. RNA was prepared from 14-day-old whole seedlings. A total of 22,560,508 WT and 24,383,585 fpa-7 reads that map polyadenylated RNA 3′ ends were aligned uniquely to the most recent A. thaliana genome release (currently TAIR10). A summary of the read statistics is given in Table S1. In each genotype, the vast majority of reads mapped to 3′ untranslated regions (UTRs) of protein-coding genes (Figure 1A–B). The DRS data can be visualised using aligned reads available at www.compbio.dundee.ac.uk/polyADB/.

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