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Heat stress enhances the accumulation of polyadenylated mitochondrial transcripts in Arabidopsis thaliana.

Adamo A, Pinney JW, Kunova A, Westhead DR, Meyer P - PLoS ONE (2008)

Bottom Line: We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment.We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 3'region.As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome, these effects need to be considered when interpreting microarray data.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Sciences, University of Leeds, Leeds, United Kingdom.

ABSTRACT

Background: Polyadenylation of RNA has a decisive influence on RNA stability. Depending on the organisms or subcellular compartment, it either enhances transcript stability or targets RNAs for degradation. In plant mitochondria, polyadenylation promotes RNA degradation, and polyadenylated mitochondrial transcripts are therefore widely considered to be rare and unstable. We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment.

Methodology/principal findings: As the Columbia genome contains a complete set of mitochondrial genes, we had to identify polymorphisms to differentiate between nuclear and mitochondrial copies of a mitochondrial transcript. We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 3'region. Heat-induced enhancement of these transcripts was quickly restored during a short recovery period.

Conclusions/significance: Our results show that polyadenylated transcripts of mitochondrial origin are more stable than previously suggested, and that their steady-state levels can even be significantly enhanced under certain conditions. As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome, these effects need to be considered when interpreting microarray data.

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Related in: MedlinePlus

The Mito1 transcript derives from the mitochondrial copy.(A) Schematic map of the mitochondrial copy of rpl2, and its nuclear counterpart integrated into the Arabidopsis genome, At2g07715. Arrows depict exon ORFs, T-DNA localises the transgene insertion in SALK_143190 Nucleotides mark differences between the two sequences and the C>U editing site of rpl2. (B) Vertical bars mark the distribution of polyadenylation sites detected in cDNA copies of Mito1 transcripts isolated at 40°C or 24°C, respectively. Only four sites were found more than once. P1 and P2 show the localisation of nested primers used for the RT-PCR. Nucleotide positions are labelled with reference to the annotated 5′ end at position 1. The 3′ end of rpl2 is located at position 3793. [28] (C) RT-PCR primers including the DraI site of the nuclear Mito1 insertion At2g07715 (40 cycles) and with primers lacking the DraI site and specific for the mitochondrial Mito1 gene rpl2 (32 cycles). Both at 24°C and at 40°C, only rpl2 specific transcripts could be amplified. EF1α was used to calibrate the amount of cDNA. Genomic wild type DNA was used as positive control (+); (-) indicates control reactions at 40°C without Reverse Transcriptase treatment. (D) RT-PCR analysis of the Mito1 transcript after sequential treatment with Alkaline Phosphatase (AP), Tobacco Acid Pyrophosphatase (TAP) and Terminator exonuclease (Ter). Lack of signal reduction, compared to the untreated sample (U) after combined treatment with AP, TAP and Ter (CAP) indicates absence of 5′CAP structure. Sensitivity of the transcript to Ter treatment (P) suggests a 5′phosphate structure for the Mito1 transcript. The Elongation Factor 1 (EF1α) transcript was assayed as a control.
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pone-0002889-g002: The Mito1 transcript derives from the mitochondrial copy.(A) Schematic map of the mitochondrial copy of rpl2, and its nuclear counterpart integrated into the Arabidopsis genome, At2g07715. Arrows depict exon ORFs, T-DNA localises the transgene insertion in SALK_143190 Nucleotides mark differences between the two sequences and the C>U editing site of rpl2. (B) Vertical bars mark the distribution of polyadenylation sites detected in cDNA copies of Mito1 transcripts isolated at 40°C or 24°C, respectively. Only four sites were found more than once. P1 and P2 show the localisation of nested primers used for the RT-PCR. Nucleotide positions are labelled with reference to the annotated 5′ end at position 1. The 3′ end of rpl2 is located at position 3793. [28] (C) RT-PCR primers including the DraI site of the nuclear Mito1 insertion At2g07715 (40 cycles) and with primers lacking the DraI site and specific for the mitochondrial Mito1 gene rpl2 (32 cycles). Both at 24°C and at 40°C, only rpl2 specific transcripts could be amplified. EF1α was used to calibrate the amount of cDNA. Genomic wild type DNA was used as positive control (+); (-) indicates control reactions at 40°C without Reverse Transcriptase treatment. (D) RT-PCR analysis of the Mito1 transcript after sequential treatment with Alkaline Phosphatase (AP), Tobacco Acid Pyrophosphatase (TAP) and Terminator exonuclease (Ter). Lack of signal reduction, compared to the untreated sample (U) after combined treatment with AP, TAP and Ter (CAP) indicates absence of 5′CAP structure. Sensitivity of the transcript to Ter treatment (P) suggests a 5′phosphate structure for the Mito1 transcript. The Elongation Factor 1 (EF1α) transcript was assayed as a control.

Mentions: The high similarity between the mitochondrial genome and its genomic insertion makes it difficult to define the origin of these transcripts. We therefore used a T-DNA insertion line (SALK_143190) to tag a mitochondrial model gene in the chromosome 2 cluster, and to sequence the nuclear copy of the gene. We labelled the transcript Mito1 as it was unclear if it derived from the mitochondrial gene rpl2 or its nuclear insert At2g07715. Sequence comparison of the nuclear insert At2g07715 with the corresponding mitochondrial gene rpl2 identified three polymorphisms, one of which created a DraI site in At2g07715 that was absent in rpl2 (Fig 2A). An RT-PCR with primers that included the DraI sequence failed to amplify any product, while rpl2-specific primers amplified Mito1 specific transcripts (Fig 2C), suggesting a mitochondrial origin of Mito1 transcripts. This was confirmed when we cloned cDNAs representing the 3′ part of the Mito1 transcript, both from material harvested at 24°C and 40°C. All clones matched the mitochondrial rpl2 transcript, and all except three resembled the unspliced version of the rpl2 transcript, with its editing site [11] unchanged. Most cDNAs had different polyadenylation sites, characteristic for the variable polyadenylation of mitochondrial transcripts (Fig. 2B, Fig.S2). Nuclease treatments suggested that the polyadenylated rpl2 transcripts did not contain a CAP structure (Fig 2D), as expected for a mitochondrial transcript. All results therefore suggest that the Mito1 transcripts represent polyadenylated transcripts of the mitochondrial rpl2 gene.


Heat stress enhances the accumulation of polyadenylated mitochondrial transcripts in Arabidopsis thaliana.

Adamo A, Pinney JW, Kunova A, Westhead DR, Meyer P - PLoS ONE (2008)

The Mito1 transcript derives from the mitochondrial copy.(A) Schematic map of the mitochondrial copy of rpl2, and its nuclear counterpart integrated into the Arabidopsis genome, At2g07715. Arrows depict exon ORFs, T-DNA localises the transgene insertion in SALK_143190 Nucleotides mark differences between the two sequences and the C>U editing site of rpl2. (B) Vertical bars mark the distribution of polyadenylation sites detected in cDNA copies of Mito1 transcripts isolated at 40°C or 24°C, respectively. Only four sites were found more than once. P1 and P2 show the localisation of nested primers used for the RT-PCR. Nucleotide positions are labelled with reference to the annotated 5′ end at position 1. The 3′ end of rpl2 is located at position 3793. [28] (C) RT-PCR primers including the DraI site of the nuclear Mito1 insertion At2g07715 (40 cycles) and with primers lacking the DraI site and specific for the mitochondrial Mito1 gene rpl2 (32 cycles). Both at 24°C and at 40°C, only rpl2 specific transcripts could be amplified. EF1α was used to calibrate the amount of cDNA. Genomic wild type DNA was used as positive control (+); (-) indicates control reactions at 40°C without Reverse Transcriptase treatment. (D) RT-PCR analysis of the Mito1 transcript after sequential treatment with Alkaline Phosphatase (AP), Tobacco Acid Pyrophosphatase (TAP) and Terminator exonuclease (Ter). Lack of signal reduction, compared to the untreated sample (U) after combined treatment with AP, TAP and Ter (CAP) indicates absence of 5′CAP structure. Sensitivity of the transcript to Ter treatment (P) suggests a 5′phosphate structure for the Mito1 transcript. The Elongation Factor 1 (EF1α) transcript was assayed as a control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483354&req=5

pone-0002889-g002: The Mito1 transcript derives from the mitochondrial copy.(A) Schematic map of the mitochondrial copy of rpl2, and its nuclear counterpart integrated into the Arabidopsis genome, At2g07715. Arrows depict exon ORFs, T-DNA localises the transgene insertion in SALK_143190 Nucleotides mark differences between the two sequences and the C>U editing site of rpl2. (B) Vertical bars mark the distribution of polyadenylation sites detected in cDNA copies of Mito1 transcripts isolated at 40°C or 24°C, respectively. Only four sites were found more than once. P1 and P2 show the localisation of nested primers used for the RT-PCR. Nucleotide positions are labelled with reference to the annotated 5′ end at position 1. The 3′ end of rpl2 is located at position 3793. [28] (C) RT-PCR primers including the DraI site of the nuclear Mito1 insertion At2g07715 (40 cycles) and with primers lacking the DraI site and specific for the mitochondrial Mito1 gene rpl2 (32 cycles). Both at 24°C and at 40°C, only rpl2 specific transcripts could be amplified. EF1α was used to calibrate the amount of cDNA. Genomic wild type DNA was used as positive control (+); (-) indicates control reactions at 40°C without Reverse Transcriptase treatment. (D) RT-PCR analysis of the Mito1 transcript after sequential treatment with Alkaline Phosphatase (AP), Tobacco Acid Pyrophosphatase (TAP) and Terminator exonuclease (Ter). Lack of signal reduction, compared to the untreated sample (U) after combined treatment with AP, TAP and Ter (CAP) indicates absence of 5′CAP structure. Sensitivity of the transcript to Ter treatment (P) suggests a 5′phosphate structure for the Mito1 transcript. The Elongation Factor 1 (EF1α) transcript was assayed as a control.
Mentions: The high similarity between the mitochondrial genome and its genomic insertion makes it difficult to define the origin of these transcripts. We therefore used a T-DNA insertion line (SALK_143190) to tag a mitochondrial model gene in the chromosome 2 cluster, and to sequence the nuclear copy of the gene. We labelled the transcript Mito1 as it was unclear if it derived from the mitochondrial gene rpl2 or its nuclear insert At2g07715. Sequence comparison of the nuclear insert At2g07715 with the corresponding mitochondrial gene rpl2 identified three polymorphisms, one of which created a DraI site in At2g07715 that was absent in rpl2 (Fig 2A). An RT-PCR with primers that included the DraI sequence failed to amplify any product, while rpl2-specific primers amplified Mito1 specific transcripts (Fig 2C), suggesting a mitochondrial origin of Mito1 transcripts. This was confirmed when we cloned cDNAs representing the 3′ part of the Mito1 transcript, both from material harvested at 24°C and 40°C. All clones matched the mitochondrial rpl2 transcript, and all except three resembled the unspliced version of the rpl2 transcript, with its editing site [11] unchanged. Most cDNAs had different polyadenylation sites, characteristic for the variable polyadenylation of mitochondrial transcripts (Fig. 2B, Fig.S2). Nuclease treatments suggested that the polyadenylated rpl2 transcripts did not contain a CAP structure (Fig 2D), as expected for a mitochondrial transcript. All results therefore suggest that the Mito1 transcripts represent polyadenylated transcripts of the mitochondrial rpl2 gene.

Bottom Line: We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment.We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 3'region.As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome, these effects need to be considered when interpreting microarray data.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Sciences, University of Leeds, Leeds, United Kingdom.

ABSTRACT

Background: Polyadenylation of RNA has a decisive influence on RNA stability. Depending on the organisms or subcellular compartment, it either enhances transcript stability or targets RNAs for degradation. In plant mitochondria, polyadenylation promotes RNA degradation, and polyadenylated mitochondrial transcripts are therefore widely considered to be rare and unstable. We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment.

Methodology/principal findings: As the Columbia genome contains a complete set of mitochondrial genes, we had to identify polymorphisms to differentiate between nuclear and mitochondrial copies of a mitochondrial transcript. We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 3'region. Heat-induced enhancement of these transcripts was quickly restored during a short recovery period.

Conclusions/significance: Our results show that polyadenylated transcripts of mitochondrial origin are more stable than previously suggested, and that their steady-state levels can even be significantly enhanced under certain conditions. As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome, these effects need to be considered when interpreting microarray data.

Show MeSH
Related in: MedlinePlus