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• W2011834538 abstract "Article15 August 1997free access The steady-state level of mRNA from the Ogura cytoplasmic male sterility locus in Brassica cybrids is determined post-transcriptionally by its 3′ region Mohammed Bellaoui Mohammed Bellaoui Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Georges Pelletier Georges Pelletier Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Françoise Budar Corresponding Author Françoise Budar Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Mohammed Bellaoui Mohammed Bellaoui Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Georges Pelletier Georges Pelletier Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Françoise Budar Corresponding Author Françoise Budar Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France Search for more papers by this author Author Information Mohammed Bellaoui1, Georges Pelletier1 and Françoise Budar 1 1Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5057-5068https://doi.org/10.1093/emboj/16.16.5057 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have investigated the control of the expression of three different configurations of the mitochondrial gene orf138, whose expression is correlated with Ogura cytoplasmic male-sterility in rapeseed cybrids. These configurations, termed Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) cybrids respectively, have the same 5′ regions but different 3′ regions. The orf138 transcript from Bam4.8/18S is 10-fold more abundant than the one from Nco2.5/13S, while no orf138 transcript from Nco2.7/13F accumulates. However, transcriptional activity measurements show that the rate of transcription is equivalent for the three configurations. These results strongly suggest that the steady-state level of mRNA from the orf138 locus is determined post-transcriptionally, most likely by its 3′ region. To determine the role of these 3′ regions, we have established an in vitro decay and processing system. In the presence of rapeseed mitochondrial lysate, synthetic RNAs corresponding to the 3′ region of the Nco2.7/13F transcript are, as expected, less stable than RNAs corresponding to the 3′ regions of the Nco2.5/13S and Bam4.8/18S transcripts. We have also observed in vitro processing of synthetic RNAs at the sites corresponding to the 3′ ends of the natural mRNAs from Nco2.5/13S and Bam4.8/18S. Further analysis of the role of these 3′ regions in in vitro RNA stability should help us to better understand post-transcriptional control in plant mitochondria. Introduction In mammals and fungi, the control of the expression of the mitochondrial genome has been extensively investigated, while relatively little is known about the control of the expression of plant mitochondrial genes. Recently, however, important progress has been achieved in the identification of plant mitochondrial promoter sequences (Binder et al., 1990; Brown et al., 1991; Covello and Gray, 1991; Mulligan et al., 1991; Binder and Brennicke, 1993; Nakazono et al., 1995). In vitro transcription systems have been established and allow the identification of sequences necessary and sufficient for efficient transcription initiation (Hanic-Joyce and Gray, 1991; Rapp and Stern, 1992; Rapp et al., 1993; Binder et al., 1995). Nevertheless, previous work has demonstrated that, as in plant chloroplasts, post-transcriptional control plays an important role in the regulation of plant mitochondrial genome expression (Finnegan and Brown, 1990; Mulligan et al., 1991). Almost nothing is known about the mechanisms that control gene expression at this level. Like chloroplast transcripts, plant mitochondrial mRNAs tend to contain inverted repeat sequences in their 3′ region that can fold into stem-loop structures (Bland et al., 1986; Schuster et al., 1986; Rothenberg and Hanson, 1987; Wahleithner and Wolstenholme, 1988; Wissinger et al., 1988; Gualberto et al., 1990; Macfarlane et al., 1990; Saalaoui et al., 1990; Kaleikau et al., 1992; Liu et al., 1992). In Chlamydomonas and higher plant chloroplasts, these inverted repeats act to stabilize upstream sequences (Stern and Gruissem, 1987; Stern et al., 1989, 1991; Adams and Stern, 1990; Stern and Kindle, 1993) and interact with enzyme complexes required for 3′ end processing (Hayes et al., 1996). In plant mitochondria, the role of the 3′ untranslated region of transcripts is still unknown. Protoplast fusion is a very powerful tool for the manipulation of plant mitochondrial genomes (Belliard et al., 1979), allowing genetic recombination between mitochondrial genomes of different species and leading to new associations of sequences. In this paper, we describe studies which were conducted on Brassica napus cybrids obtained after fusion of protoplasts from plants bearing normal Brassica cytoplasm and plants bearing Ogura radish cytoplasmic male sterility (CMS)-inducing cytoplasm (Bannerot et al., 1977; Pelletier et al., 1983). CMS is a maternally inherited, mitochondrially encoded trait that prevents the production of functional pollen but maintains female fertility (Vedel et al., 1994). The comparison between the closely related mitochondrial genomes of male-sterile (13S) and fertile revertant (13F) progenies of a cybrid allowed the identification of a sequence (a 2.5 kb NcoI fragment termed Nco2.5/13S) associated with Ogura CMS (Bonhomme et al., 1991). Two open reading frames (orf138 and orfB) were found in this fragment, and orf138 expression was correlated with Ogura CMS in rapeseed cybrids (Bonhomme et al., 1992). Grelon et al. (1994) demonstrated that orf138 is translated into a mitochondrial membrane protein in sterile cybrids. The fertile revertant cybrid 13F contains the orf138 gene on the Nco2.7/13F fragment; no transcripts from this gene accumulate and the orf138 protein is not detectable (Bonhomme et al., 1992; Grelon et al. 1994). A third configuration of the orf138 gene (Bam4.8/18S) was found in another sterile cybrid, termed 18S (Grelon et al., 1994). Previous Northern blot analyses have shown differences in the steady-state orf138 mRNA accumulation between the three configurations Nco2.5/13S, Nco2.7/13F and Bam4.8/18S specific to the 13S, 13F and 18S cybrids respectively (Grelon et al., 1994). The three configurations of the mitochondrial gene orf138 have been shown to have the same 5′ region but different 3′ regions (Figure 1). The differences between these three configurations make these cybrids ideal material for examining the role of 3′ untranslated regions in plant mitochondrial genome expression. Figure 1.Restriction maps of the three configurations Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) cybrids respectively. The sequences of the Nco2.7/13F and Bam4.8/18S fragments which are common with the Nco2.5/13S fragment are indicated by a thick black line. White box: trnfM gene coding for initiator methionine tRNA; light grey box: orf138 gene; dark grey box: orfB gene; striped box: atpA gene. In vivo accumulated transcripts from each configuration are indicated by the bent arrows. Download figure Download PowerPoint In this report we show that the expression of the genes at the CMS locus is determined post-transcriptionally. Our data suggest that the 3′ region of the orf138 transcripts plays an important role in transcript processing and stability. Results Expression of the orf138 gene in Brassica cybrids Steady-state level of orf138 mRNA. To quantify the differences in the steady-state orf138 mRNA accumulation between the three configurations Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, slot blots were carried out with controlled quantities of total RNA from the three cybrids. Hybridization with an orf138 probe showed that the orf138 transcript from Bam4.8/18S in sterile cybrid 18S is 10-fold more accumulated than the one from Nco2.5/13S in sterile cybrid 13S, whilst no orf138 transcript is detected from Nco2.7/13F in cybrid 13F (Figure 2A). To determine whether these differences in gene expression are due to differences in mitochondrial DNA copy number, orf138 abundance in total DNA was quantified on a slot blot carrying total DNA. Figure 2B shows that the ratio between orf138 and atpA signals is approximately identical for the three cybrids. The orf138 locus is, therefore, present in an equal number of copies in the three cybrids. Thus differential amplification of the orf138 locus in the three cybrids cannot account for the differences in steady-state accumulation of orf138 mRNA. Figure 2.Differential abundance of the orf138 mRNA is not accompanied by differences in mitochondrial DNA copy number in Brassica cybrids. Slot blots of total RNA (A) or total DNA (B) were hybridized successively with radiolabelled orf138 and atpA probes (atpA was used as an internal control). Each lane contains decreasing quantities (10 μg, 1 μg, 100 ng) of total RNA or total DNA of 13S, 13F or 18S cybrids. Download figure Download PowerPoint A transcript is synthesized from Nco2.7/13F but it does not accumulate. To check for the presence of an orf138 transcript from Nco2.7/13F, RT-PCR experiments were performed on RNA from the 13F cybrid (Figure 3). An amplified fragment of the expected size (1100 bp) was obtained when cDNA from the 13F cybrid was used as template (+RT). This fragment was not obtained when the 13F RNA sample was not previously incubated with reverse transcriptase (−RT). Cloning and sequencing of the PCR product revealed that the 1100 bp fragment contains the Nco2.7/13F sequence and thus is derived from an orf138 transcript. This sensitive experiment shows that an orf138 transcript is synthesized from Nco2.7/13F, although it is not detectable on Northern or slot blots, and that orf138 is co-transcribed with atpA in this configuration. Figure 3.An orf138-atpA co-transcript is synthesized from the Nco2.7/13F configuration. RT-PCR experiment carried out using the two primers A and B, shown in the lower panel of the figure on the restriction map of Nco2.7/13F. The substrate was total RNA from buds of the 13F cybrid following reverse transcription (+RT) or without reverse transcription (−RT). The size of the PCR amplified fragment with the reverse transcribed sample is indicated. Lane M contains marker fragments (1 kb ladder BRL). Download figure Download PowerPoint Nco2.5/13S, Nco2.7/13F and Bam4.8/18S are transcribed at the same rate. Since the three configurations Nco2.5/13S, Nco2.7/13F and Bam4.8/18S have the same 5′ region, it is likely that these configurations have identical promoters and thus could be transcribed at equal rates. To test this hypothesis, run-on experiments were used to compare the transcription of orf138 in the three configurations (Figure 4). Isolated mitochondria from 13S, 13F and 18S cybrids were incubated with radiolabelled nucleotides. The run-on products, which reflect the elongation of pre-existing transcriptional complexes (Mulligan et al., 1991), were hybridized to slot blots carrying the orf138 and atp9 gene sequences. Quantitation analysis by phosphorimaging indicated that the ratio between orf138 and atp9 signals is approximately identical for the three cybrids. The orf138 gene has, therefore, a similar rate of transcription in the three cytotypes. Thus, differential gene expression must reflect post-transcriptional events. Figure 4.The orf138 gene is transcribed at the same rate in the three configurations. Run-on transcription products synthesized by mitochondria from 13S, 13F or 18S were hybridized to slot blots carrying 4 μg of orf138, atp9 (used as an internal control) and pBluescript SK− plasmid (used as negative control). After hybridization, the membranes were treated with RNase, washed and then autoradiographed. Download figure Download PowerPoint Analysis of the 5′ and 3′ termini of the orf138 transcripts Determination of the 5′ end of the orf138 transcripts. Primer extension experiments were used to map the 5′ ends of the transcripts from Bam4.8/18S and Nco2.5/13S (Figure 5A). Using total RNA from 18S and 13S as templates, three primer extension products were obtained. This experiment showed that the orf138 transcripts from Bam4.8/18S (in 18S mitochondrial RNA) and Nco2.5/13S (in 13S mitochondrial RNA) have identical 5′ termini. A sequence reaction using a subclone of the orf138 locus and the primer used in primer extension allowed the precise location of the 5′ termini at the C, G and U nucleotides 107, 106 and 105 nucleotides respectively from the initiation AUG of the orf138 coding region. Altough we did not obtain primer extension products for the 13F cybrid, we assume that the orf138 transcripts from Nco2.7/13F have the same 5′ termini. These 5′ termini are 3, 4 and 5 bp upstream of the 5′ end previously suggested for the Nco2.5/13S transcript (Bonhomme et al., 1992). Since we used a sequence ladder to size our primer extension products, we suggest that our mapping is more accurate. Figure 5.The 5′ termini of the orf138 transcripts are identical in cybrids 13S and 18S, and are processing sites. (A) 5′ end mapping of the orf138 transcripts. Labelled oligonucleotide PE, indicated below the figure on the restriction map of the 5′ region of the orf138 locus, was hybridized to total RNA of 13S, 13F and 18S. After reverse transcription, the primer extension products were separated on a 6% sequencing gel next to sequencing reactions using PE as primer and the 5′ region of the orf138 locus (subcloned in Bluescript) as template. The sequence surrounding the 5′ ends is given on the right. The arrows correspond to the 5′ ends and the boxed sequence corresponds to the end of the trnfM sequence. (B) Hybridization of phosphorylated or capped mitochondrial RNA from the 18S cybrid. After hybridization of labelled mitochondrial RNA to a Southern blot of the HincII-XbaI subclone, the blots were treated with RNase, washed and autoradiographed. The labelled mitochondrial RNA used in each case is indicated above each panel. Download figure Download PowerPoint Figure 6.Analysis of the 3′ termini of the orf138 transcript of Bam4.8/18S. (A) RNase protection of the 3′ termini of the orf138 transcript. Autoradiograph of RNase mapping experiments using 20 μg of total RNA isolated from 18S (1 and 2). The control lane corresponds to protection in the presence of 26 μg of yeast tRNA (3). The probe derived by in vitro transcription of the EcoRV-EcoRV fragment has been loaded (P) and is indicated below the figure on the restriction map of Bam4.8/18S. The sizes of the protected fragments are indicated. (B) Potential secondary structure formed around the mapped 3′ termini of the orf138 transcript from Bam4.8/18S. The 3′ ends are indicated by arrows. (C) Northern blot analysis. Mitochondrial RNA was extracted from 18S cybrid, fractionated by electrophoresis, and used for Northern blot analysis. PCR amplified probes are specific to the 3′ untranslated region of orf138 transcript from Bam4.8/18S just upstream of the major 3′ end (lane 1) or to the t-element (lane 2). Download figure Download PowerPoint To determine if the 5′ ends of the orf138 transcripts are processing sites or transcription initiation sites, in vitro-capped mitochondrial RNA and 5′-phosphorylated mitochondrial RNA were hybridized to restriction fragments of the cloned 5′ region of the orf138 locus. The capping enzyme guanylyltransferase specifically labels the 5′ di- or triphosphate termini of primary transcripts (Christianson and Rabinowitz, 1983), while the polynucleotide kinase enzyme labels the monophosphate 5′ ends formed by RNA processing. The HincII-XbaI fragment (325 bp) containing the 5′ ends of the orf138 transcripts was labelled after hybridization with phosphorylated mitochondrial RNA, but was not labelled after hybridization with capped RNA (Figure 5B). The capped RNA used in this experiment was also hybridized to an orfB fragment and gave a similar result to the experiment shown in Figure 7C (data not shown), showing that the capping of the mitochondrial RNA was successful. Thus, the 5′ ends of the orf138 transcripts are produced by a processing event, and transcription of the orf138 locus probably initiates upstream of the adjacent trnfM gene. This result was expected because the flanking sequences of the 5′ end of orf138 transcript show no similarity to the dicot consensus sequence for transcription initiation (Brown et al., 1991; Binder and Brennicke, 1993). Figure 7.The 5′ end of the atpA transcript from Nco2.7/13F is a processing site rather than a transcription initiation site. (A) Restriction maps of Nco2.2 and Nco2.7/13F. The transcripts detected from each configuration are indicated by the bent arrows. The regions of the orfB and atpA loci used as probes in RNase mapping (Map) and in vitro capping (Cap) analysis are shown by thick lines below each diagram. (B) atpA 5′ end analysis: (Map) RNase mapping of the 5′ ends of the atpA transcripts. The region from the orf138 coding region to the initiation codon of the atpA gene from Nco2.7/13F was used as probe (P). M: RNase protection using mitochondrial RNA isolated from 13F cybrid. Cap: RNase protection analysis of capped mitochondrial RNA. Mitochondrial RNA from 13F was radiolabelled by capping and hybridized with the unlabelled probe. After treatment of the heteroduplexes with RNase, the protected fragments were loaded. The DNA sequence arround the 5′ end of atpA gene is shown below and the 5′ end corresponding to the processing site is represented by a vertical arrow. (C) Positive control (orfB 5′ end analysis): (Map) RNase mapping of the 5′ ends of the orfB transcripts. The transcript derived by in vitro transcription of the HindIII-EcoRI fragment from orfB locus was used as probe (P). M: RNase protection using mitochondrial RNA isolated from 13F cybrid. The 5′ ends of the orfB transcript are indicated by arrows. Cap: RNase protection analysis of capped mitochondrial RNA. In vitro-capped mitochondrial RNA from 13F cybrid which is used in the capping experiment with atpA transcript, was hybridized with the unlabelled probe from the orfB locus. After treatment of the heteroduplexes with RNase, the protected fragments were loaded on the gel. The DNA sequence at the transcription initiation sites is shown below. Transcription initiation sites are presented by bent arrows and the highly conserved sequence in the dicot promoter regions (Brown et al., 1991; Binder and Brennicke, 1993) is boxed. Download figure Download PowerPoint A tRNA-like element is present immediately downstream of the 3′ ends of the orf138 transcript from Bam4.8/18S. In order to identify the 3′ end of the orf138 transcript in Bam4.8/18S, an antisense probe was used in RNase mapping experiments (Figure 6A). Two ladders of several bands were obtained from 18S mitochondrial RNAs. For each ladder, two major protected fragments were seen (91-94 and 73-74 nucleotides). After a long exposure of the gel, another protected fragment (164 nucleotides) was seen which may represent a minor 3′ end. Using the MFOLD software in the GCG package, a tRNA-like structure can be formed by folding the 3′ untranslated region of the Bam4.8/18S (Figure 6B). Similar tRNA-like structures have been identified in wheat mitochondrial DNA and have been termed ‘t-elements’ (Hanic-Joyce et al., 1990). Interestingly, the t-element sequence is identical in size to the smallest protected product obtained in the RNase protection experiments (Figure 6A). Thus, the 73-74 protected fragments probably reflect RNase protection of the processed t-element rather than protection by the 3′ untranslated region of the orf138 transcripts from Bam4.8/18S. The minor protection fragment is the right size to represent an orf138 transcript including the t-element; the major 3′ end corresponds to orf138 transcripts lacking the t-element. This interpretation is confirmed by Northern experiments. A probe complementary to the t-element does not hybridize to orf138 transcripts, but does recognize a tRNA-sized transcript in mitochondrial RNA of plants carrying the Bam4.8/18S fragment (Figure 6C). A processing event between the orf138 and atpA coding regions is responsible for the formation of the 3′ end of the orf138 transcript from Nco2.7/13F. RT-PCR experiments have shown that a co-transcript orf138-atpA is synthesized from Nco2.7/13F (Figure 3), and primer extension experiments revealed that the 5′ end of the atpA transcript is localized 199 nucleotides upstream of the initiation AUG of the atpA coding region (data not shown). To check the hypothesis that the monocistronic atpA transcript is formed by RNA processing of the orf138-atpA co-transcript, the nature of the 5′ end of the atpA transcript was determined by an in vitro capping experiment. Total mitochondrial RNA from the 13F cybrid was labelled by in vitro capping and hybridized to the unlabelled antisense probe specific to the 5′ region of the atpA gene (Figure 7A). The duplexes were treated with RNases and the protected fragments were loaded on a sequencing gel (Figure 7B). No labelled RNA fragments corresponding to the 5′ terminus of the atpA transcript were obtained. Thus, the 5′ end of the atpA transcript was not labelled with the capping enzyme guanylyltransferase. This result was expected because the flanking sequences of the 5′ end of atpA transcript show no similarity to the dicot consensus sequence for transcription initiation (Brown et al., 1991; Binder and Brennicke, 1993). We assume, therefore, that the 3′ end of the unstable orf138 transcript from Nco2.7/13F is produced by processing at the 5′ end of the atpA transcript. The Brassica orfB transcript was used as a positive control for the in vitro capping experiments (Figure 7B). When the unlabelled antisense probe derived by in vitro transcription of the HindIII-EcoRI fragment from the orfB locus was hybridized to the same in vitro capped RNA, two fragments of ∼279 and 274 bp were protected and correspond to the 5′ termini of the orfB transcript predicted by 5′ RNase mapping, showing that the capping of mitochondrial RNA was successful. Analysis of the 3′ terminus of the orf138 transcript from Nco2.5/13S. Previous RNase protection mapping of the 3′ end of the orf138 transcripts from Nco2.5/13S showed that the 3′ end mapped two nucleotides downstream of a potential stem-loop structure (Bonhomme et al., 1992). However, in this previous work, the sizes of the protected fragments were estimated using a DNA ladder and by assuming that the RNA fragments migrate 5-10% slower than the DNA fragments of the same size. Because the precise location of the 3′ ends is important for the decay and processing experiments described below, the 3′ terminus was remapped by co-electrophoresis of the RNase-protected fragments with an RNA ladder (data not shown). This showed that the true 3′ end lies 20 nucleotides downstream of the potential stem-loop structure (Figure 8). Figure 8.Potential secondary structure formed upstream of the 3′ end of the orf138 transcript from Nco2.5/13S. The 3′ end is indicated by an arrow. Download figure Download PowerPoint In vitro decay and processing analysis of the 3′ region of orf138 transcripts A mitochondrial lysate processes synthetic RNAs corresponding to the 3′ region of the Nco2.5/13S and Bam4.8/18S transcripts. To determine the role of secondary structures found in the 3′ region of orf138 transcripts, an in vitro decay and processing system was established. Labelled transcripts containing the 3′ region and downstream sequences of orf138 transcripts were incubated with mitochondrial lysate for 0-45 min as described in Materials and methods. An RNA precursor of 508 nucleotides corresponding to the 3′ region of the Nco2.5/13S transcript is rapidly converted into 355- and 153-nucleotide fragments (Figure 9A). Thus, the 3′ region of the orf138 transcript from Nco2.5/13S is a substrate for rapid endonucleolytic cleavage in rapeseed mitochondrial lysate. To determine whether the cleavage occurred downstream of the stem-loop structure shown in Figure 8, a second synthetic RNA containing the same upstream sequences but a shorter downstream 3′ extension (27 nucleotides) was used in in vitro processing. As shown in Figure 9A, the 388 nucleotide substrate is converted to a 355 nucleotide fragment identical to that formed by cleavage of the 508 nucleotide substrate. This strongly suggests that the endonucleolytic cleavage occurs downstream of the stem-loop structure of the Nco2.5/13S transcript. A synthetic RNA terminating at this presumed endonucleolytic site co-migrates with the in vitro-processed RNA (Figure 9B). Even if the incubation time was reduced to 30 s, no intermediate form with a short 3′ extension in comparison with the mature product was seen (Figure 9B). These data suggest that the 3′ end of the orf138 transcript from Nco2.5/13S is produced by endonucleolytic cleavage. The 3′ end of the in vitro processing product is calculated to be 3 bp upstream of our estimation of the mature in vivo 3′ end. Thus we suggest that the 3′ end of the in vitro processing product is similar to the mature in vivo 3′ end, and given the limits to the resolution of the techniques employed, may well be identical to it. Figure 9.In vitro processing of precursor RNAs containing the 3′ region and downstream sequences of orf138 transcripts. Labelled transcripts were incubated with rapeseed mitochondrial lysates for 0, 5, 15 and 45 min. The labelled RNA used in each case is indicated below the figure on the restriction map. Times are shown above each panel. The molecular weights are estimated from the migration of in vitro-generated transcripts of known size (lane M). To the left are the deduced structures of the precursor and the major processed products. (A) The precursor RNA was a 508- or a 388-nucleotide transcript corresponding to the 3′ region of orf138 transcripts from Nco2.5/13S (the 388 nucleotide transcript corresponds to the 5′ terminal sequence of the 508 transcript). (B) Lane 1: synthetic RNA terminating at the presumed processing site without incubation with protein; lanes 2, 3, 4 and 5: the 508 nucleotide RNA corresponding to the 3′ region of orf138 transcripts from Nco2.5/13S incubated with mitochondrial lysate for 0 s, 30 s, 2 min and 20 min respectively. (C) The precursor RNA is a 278-nucleotide transcript which corresponds to the 3′ region of orf138 transcripts from Bam4.8/18S. (D) The precursor RNA is a 414 nucleotide which corresponds to the 3′ region of orf138 transcripts from Nco2.7/13F. Download figure Download PowerPoint To determine if the t-element which is present immediately downstream of the 3′ end of the Bam4.8/18S transcript (Figure 6B) is processed in vitro, a synthetic RNA containing the 3′ region of the Bam4.8/18S transcripts and the t-element sequence not present in the mature transcript was incubated in the mitochondrial lysate. Three major processed products of ∼143 , 75 and 60 nucleotides were observed (Figure 9C). These sizes are consistent with the predicted molecules which would be obtained by cleavage at the presumed in vivo 3′ end of the Bam4.8/18S transcripts and downstream of the t-element sequence. These results are entirely consistent with the RNase protection data with the natural transcripts. Our results suggest that the structures found at the 3′ region of the Nco2.5/13S and Bam4.8/18S transcripts act as efficient processing signals that generate the 3′ ends found in vivo. However, we have not observed in vitro processing at the 3′ end of the orf138 transcripts from Nco2.7/13F. The rapid degradation of RNA corresponding to the intergenic region between orf138 and atpA may be responsible for the failure to observe a processed product (Figure 9D). In vitro decay of the 3′ region of orf138 transcripts. To test whether the different 3′ regions of the orf138 transcripts show differential RNA stability in an in vitro decay system, synthetic RNAs of the 3′ region of orf138 transcripts terminating at the mature in vivo 3′ ends were incubated with mitochondrial lysates for 0-60 min. The kinetics for the decay of the three 3′ regions show that the most rapidly decaying RNA is, as expected, the 3′ region of the Nco2.7/13F transcript. The most stable RNA is, as expected, the 3′ region of the Bam4.8/18S transcript. The 3′ region of the Nco2.5/13S transcript was moderately degraded (Figure 10). Thus, it is probable that the differences in the steady-state level of orf138 mRNA between the three configurations involve the divergent 3′ regions which play important roles in RNA stability. However, the rate of the degradation of the 3′ region of the Nco2.7/13F transcript is not as fast as would be predicted by the rarity of orf138 transcripts from Nco2.7/13F in vivo. It is possible tha" @default.
• W2011834538 created "2016-06-24" @default.
• W2011834538 creator A5024490042 @default.
• W2011834538 date "1997-08-15" @default.
• W2011834538 modified "2023-09-27" @default.
• W2011834538 title "The steady-state level of mRNA from the Ogura cytoplasmic male sterility locus in Brassica cybrids is determined post-transcriptionally by its 3' region" @default.
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• W2011834538 doi "https://doi.org/10.1093/emboj/16.16.5057" @default.
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