Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5' and 3' end formation

Abstract

With CR-RT-PCR as primary approach we mapped the 5' and 3' transcript ends of all mitochondrial protein-coding genes in Arabidopsis thaliana. Almost all transcripts analyzed have single major 3' termini, while multiple 5' ends were found for several genes. Some of the identified 5' ends map within promoter motifs suggesting these ends to be derived from transcription initiation while the majority of the 5' termini seems to be generated post-transcriptionally. Assignment of the extremities of 5' leader RNAs revealed clear evidence for an endonucleolytic generation of the major cox1 and atp9 5' mRNA ends. tRNA-like structures, so-called t-elements, are associated either with 5' or with 3' termini of several mRNAs. These secondary structures most likely act as cis-signals for endonucleolytic cleavages by RNase Z and/or RNase P. Since no conserved sequence motif is evident at post-transcriptionally derived ends, we suggest t-elements, stem-loops and probably complex higher order structures as cis-elements for processing. This analysis provides novel insights into 5' and 3' end formation of mRNAs. In addition, the complete transcript map is a substantial and important basis for future studies of gene expression in mitochondria of higher plants.

Figure 1.

CT–RT–PCR analysis of mitochondrial mRNAs. Two different CR–RT–PCR strategies were followed in the transcript analysis of the 32 protein-coding genes encoded in the mitochondrial genome of A. thaliana. The main difference is that the approach outlined in (A) includes two PCRs and cloning with subsequent sequence analysis of individual clones while in the alternative approach (B) PCR products obtained by a single PCR step were directly sequenced. Further explanations see in the text. FP: Forward primer; RP: reverse primer; PcDNA: oligonucleotide used to prime cDNA synthesis.

Figure 2.

Mapping of cox1 transcript ends. (A) The cox1 reading frame (grey box) is flanked by 10 and 17-kb non-coding DNA (indicated by dotted arrows), respectively, from the next genes (white boxes). The location of the primers is depicted by black arrows. The probe used in the northern analysis is given as a black bar (+24 to +1,524). (B) cox1 transcript analysis using the experimental strategy depicted in Figure 1A. Products obtained in the first PCR performed with primer pair Atcox1-1/Atcox1-3 were separated on a 1% agarose gel (lane 1). cDNA fragments with sizes of about 650, 500 and 400 bp (corresponding to products A, B and C indicated in the right margin of lane 1) were used as DNA templates in the second PCR, which was carried out with primer pair Atcox1-2/Atcox1-4. The products were separated in lanes A–C (designation of the lanes corresponds to the products of the first PCR used as templates). (C) CR–RT–PCR analysis following the single PCR approach (outlined in Figure 1B) using oligonucleotide Atcox1-1 for cDNA synthesis and primer pair Atcox1-2/Atcox1-4 for amplification. Similar cDNA fragments were obtained from total RNA (t) and mitochondrial RNA (lane mt). Sizes of DNA marker fragments (lanes M) are given in kilobases. (D) Sequence chromatogram of a cDNA fragment representing the cox1 steady state mRNA pool. Nucleotide identities are only shown for the predominant sequence. The corresponding sequences up to the 5′ as well as the 3′ end of the cDNA are given. Further explanations are given in the text.

Figure 3.

Primer extension and northern blot analysis of cox1 transcripts. (A) Extension products generated in a reverse transcription reaction from oligonucleotide Atcox1-2 (Figure 2A) are separated along DNA marker fragments. A major product (black arrow) detects a 5′ end about 240 nucleotides upstream of the ATG, which is consistent with the major 5′ terminus mapped in the CR–RT–PCR analysis. (B) The major end(s) is (are) also detected in the extension reaction with oligonucleotide Atcox1-5. The corresponding products (lane 1–5) were separated together with sequencing reaction products (lanes G, A, T and C) obtained with the same primer. These ends (black arrows) scatter over three nucleotides with the most prominent end found at −241 and minor termini at −240 and −239. The sequence is given in both orientations. (C) Northern blot analysis performed with a probe covering most of the cox1 reading frame as indicated in Figure 2A. A single mRNA of about 1900 nucleotides is detected in total (t) and mitochondrial (mt) RNA preparations from A. thaliana cell suspension culture of ecotype Col.

Figure 4.

Stem–loop structures found directly upstream of the mapped 3′ ends. The termini are indicated by arrows.

Figure 5.

Sequences found conserved at the 5′ termini of the 26S rRNA as well as atp9 and nad6 mRNAs. The 5′ terminal nucleotides are underlined. Differing nucleotide identities are indicated with lower case letters in italics.

Figure 6.

The 5′ terminus of the cox1 steady state mRNA is generated by an endonucleolytic cleavage. (A) The cox1 gene (gray box) is transcribed from a promoter (bent arrow) located 355 nucleotides upstream of the ATG. The precursor RNA (dotted line) is processed by endonucleolytic cleavage (scissor) into a mature cox1 mRNA and a small 5′ leader RNA. In an RNA ligation reaction (bottom line) endogenous mitochondrial 5S rRNA (black bar) is ligated to the cox1 5′ leader RNA and then used as anchor in the cDNA synthesis initiated at primer At5S-5 and a subsequent PCR with primer At5S-mega.R and Atcox1-lm.H. (B) PCR products (lane P) obtained in the reaction described earlier are separated on an agarose gel alongside DNA marker fragments (lane M). A single 200-bp product is obtained which contains the 5S rRNA ligated to the 3′ end of the potential cox1 5′ leader fragment. (C) The leader sequence upstream of the mature cox1 5′ end can be folded into a tRNA-like structure forming a perfectly conserved ψ-uridine arm. The 5′ end of this t-element was also mapped in a CR–RT–PCR analysis using 5S rRNA as anchor (Supplementary Figure 31).

Figure 7.

In the A. thaliana mitochondrial genome several t-elements can be identified. Either the 5′ or 3′ ends of these t-elements coincide with 5′ and/or 3′ termini of mitochondrial mRNAs. This suggests that these t-elements direct cleavage of the respective precursor RNAs by RNase Z and RNase P in vivo.

Figure 8.

Stem–loop structures found at 5′ mRNA ends. Like the t-elements the ends of the stem–loop structures coincide with mRNA termini. These stem–loop structures might thus mimic tRNA acceptor stems and direct cleavage by RNase Z or RNase P.