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. 2014;11(7):968-76.
doi: 10.4161/rna.29781. Epub 2014 Jul 29.

RNA processing factor 7 and polynucleotide phosphorylase are necessary for processing and stability of nad2 mRNA in Arabidopsis mitochondria

Birgit Stoll  1 , Daniel Zendler  1 , Stefan Binder  1
Affiliations

RNA processing factor 7 and polynucleotide phosphorylase are necessary for processing and stability of nad2 mRNA in Arabidopsis mitochondria

Birgit Stoll et al. RNA Biol. 2014.

Abstract

Post-transcriptional maturation of plant mitochondrial transcripts requires several steps. Among these, the generation of mature 5' ends is still one of the most enigmatic processes. Toward a characterization of proteins involved in 5' processing of mitochondrial transcripts in Arabidopsis (Arabidopsis thaliana), we now analyzed 5' maturation of nad2 transcripts. Based on natural genetic variation affecting 5' ends of nad2 transcripts in ecotype Can-0 and complementation studies we now identified RNA processing factor 7, which takes part in the generation of the 5' terminus of the mature nad2 mRNA. RPF7 is a relatively short regular P-class pentatricopeptide repeat protein comprising seven canonical P repeats and a single short S repeat. The corresponding allele in Can-0 encodes a truncated version of this protein lacking two C-terminal repeats, which are essential for the function of RPF7. Furthermore we established transgenic plants expressing artifical microRNAs targeting the mitochondrial polynucleotide phosphorylase (PNPase), which results in substantial reduction of the PNPase mRNA levels and strong knockdown of this gene. Detailed quantitative studies of 5' and 3' extended nad2 precursor RNAs in these knockdown plants as well as in the rpf7-1 knockout mutant suggest that 5' processing contributes to the stability of mitochondrial transcripts in plants.

Keywords: 5′ end processing; RNA; RNA processing factor; arabidopsis thaliana; mitochondria; pentatricopeptide repeat proteins; polynucleotide phosphorylase.

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Figures

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Figure 1. In Arabidopsis accessions Can-0 and Chat-1 nad2 precursor RNAs accumulate to higher levels. (A) Schema of the nad2 reading frame (gray box). 5′ (bent arrows) and 3′ ends (pinhead) are indicated, their positions are given relative to the translation start codon (5′ end; NATG, n = -1) or to the translation stop codon (3′ end; TAAN, n = +1). Locations of the northern blot hybridization probes are indicated by black lines. Primers used for nad2 transcript analysis are represented by horizontal arrows. (B) CR-RT-PCR analysis of nad2 RNAs with primer pair Atnad2ab-2/Atnad2cde-3. Accessions are given above the image. Sizes of DNA marker fragments are indicated in the left margin. PCR products are marked on the right hand side (product 1, 340 bp; product 2, 510 bp; product 3, 620 bp). This figure has been assembled from different images. (C) northern blot hybridization with probe a representing parts of the nad2 reading frame. Positions of the cytoplasmic 26S and 18S rRNAs are marked. Stained 26S rRNA was used as loading control (bottom part). Prominent transcripts are marked on the right hand side (transcript 1, ~1,650 nucleotides (nt); transcript 2, ~1,950 nt; transcript 3, ~3,000 nt).
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Figure 2. The Col allele of At2g28050 (RPF7) restores the efficient 5′ maturation of the nad2 mRNA in Can-0, Chat-1 and in the rpf7–1 mutant (SALK_058434). (A) CR-RT-PCR analysis of nad2 transcripts in Can-0, the rpf7–1 mutant (left panel) and Chat-1 (right panel) and in the same plant lines transformed with the Col allele of RFF7 (+RPF7 (Col)). (B) northern blot hybridization performed with a probe complementary to the sequence of the open reading frame (probe a, indicated in Figure 1). Labeling is identical to Figure 1.
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Figure 3. Sequences and structures of RPF7 proteins from different Arabidopsis accessions. (A) Amino acid sequences are given in the one letter code. Divergent amino acids are given in bold and underlined letters. A premature translation stop codon is represented by an asterisk (*). The deletion in Chat-1 is indicated by a dashed line. Crucial positions (3, 6 and 1’) are given in black. The pentatricopeptide repeats are given according to a previous study above the sequence. (B) Cartoons demonstrating the structures of the distinct RPF7 proteins from the different accessions (indicated on the right hand side). Light gray boxes represent canonical P motifs (P1–7) whereas an S repeat is given as dark gray box. Incomplete motifs are marked with one (C-terminal truncated: P3‘,6‘) or two primes (N-terminal truncated: P7“).
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Figure 4. Accumulation of large nad2 transcripts in amiR-PNP-3 plants. Northern blot analyses of PNPase knockdown, Col wild-type, Can-0 wild-type, rpf7–1 plants, as well as Can-0 wild-type and rpf7–1 lines complemented with the RPF7 allele from Col (+RPF7 (Col)). Probes used in these analyses are indicated in Figure 1 and represent the nad2 reading frame (A), sequences upstream of the mature 5′ end (-122) (B) and the region downstream of the mature 3′ terminus (C). All hybridizations were done with the same membrane. Labeling corresponds to Figure 1. Loading of the RNA gels was checked by a methylene blue staining of cytoplasmic rRNAs on the membrane (26S rRNA).
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Figure 5. Quantitative analysis of nad2 transcripts in distinct plant lines. Real-Time qRT-PCR of nad2 RNAs covering the reading frame (A), 5′ region (B) and 3′ region (C). The nad2 transcript levels are given relative to the level in Col wild type, which was arbitrarily set to 1.
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