Multiple checkpoints for the expression of the chloroplast-encoded splicing factor MatK

Abstract

The chloroplast genome of land plants contains only a single gene for a splicing factor, Maturase K (MatK). To better understand the regulation of matK gene expression, we quantitatively investigated the expression of matK across tobacco (Nicotiana tabacum) development at the transcriptional, posttranscriptional, and protein levels. We observed striking discrepancies of MatK protein and matK messenger RNA levels in young tissue, suggestive of translational regulation or altered protein stability. We furthermore found increased matK messenger RNA stability in mature tissue, while other chloroplast RNAs tested showed little changes. Finally, we quantitatively measured MatK-intron interactions and found selective changes in the interaction of MatK with specific introns during plant development. This is evidence for a direct role of MatK in the regulation of chloroplast gene expression via splicing. We furthermore modeled a simplified matK gene expression network mathematically. The model reflects our experimental data and suggests future experimental perturbations to pinpoint regulatory checkpoints.

Figure 1.

RNA gel-blot analysis of matK across tobacco development. Total RNA (5 μg per lane) from seedlings of different ages (dpi) and from leaves of older plants was extracted, separated on an agarose gel, blotted to a nylon membrane, and hybridized with different radioactive probes. RNA gel-blot analyses with an exon and an intron probe were carried out. Bands that could be identified as unspliced precursor RNAs (p), free intron (i), and mature, spliced RNA (m) are indicated on the left. nt, Nucleotides. Probe positions for each autoradiogram are indicated on the right. The labeled bands were densitometrically quantified on a phosphorimager. Values were normalized to the top value of all seedling samples, which was arbitrarily set to 1. These values are displayed in the chart shown at top (black graph = precursor RNA; gray graph = free intron; dashed graph = mature tRNA). The experiment was replicated with independent RNA samples (data not shown), which allowed the determination of sd values shown here. As a loading control, blots were stained with methylene blue prior to hybridization; this is shown below the autoradiograms. Protein values (dotted line) are taken from western analyses published previously (Zoschke et al., 2010). Arrows indicate the largest discrepancies in the dynamics of protein and mRNA levels. [See online article for color version of this figure.]

Figure 2.

Run-on transcription analysis of all intron-containing genes in the chloroplast and additional controls. A, RNA was extracted from run-on reactions, which were carried out with chloroplasts purified from 7- and 25-d-old plants and hybridized on macroarrays. The array comprised probes for all chloroplast introns (In) plus additional controls, including a negative control (pBlue., pBluescript II SK+). Probes were spotted as duplicates (only one spot shown). The signals shown here are examples of two biological replicates. The data were used to generate the charts in B. For better visualization, different exposure times are shown for different probes (dashed line = short; gray lines = intermediate; black lines = long). B, Comparison of transcription rates and RNA steady-state levels in 25- versus 7-d-old material. mRNA levels were assayed by northern blot and quantified from two independent experiments (gray bars). Transcription rates were assayed by run-on analysis and quantified from two independent assays (black bars; see A). Note the logarithmic scale of the y axis. Assayed genes are indicated below. sd values were derived from two biological replicates.

Figure 3.

RNA gel-blot analysis of MatK target RNAs and trnG across tobacco development. Total RNA (5 μg per lane) from seedlings of different ages (dpi) and from leaves of older plants were extracted, separated on an agarose gel, blotted to a nylon membrane, and hybridized with different radioactive probes. Each panel includes two RNA gel-blot analyses with an exon and an intron probe. Bands that could be identified as unspliced precursor RNAs (p), free intron (i), and mature, spliced RNA (m) are indicated on the left. nt, Nucleotides. Probe positions for each autoradiogram are indicated on the right. The labeled bands were densitometrically quantified on a phosphorimager. Values were normalized to the top value of all seedling samples, which was arbitrarily set to 1. These values are displayed in the charts shown at the top of each panel (black graphs = precursor RNAs; gray graphs = free introns; dashed graphs = mature RNAs). The experiment was replicated with independent RNA samples (data not shown), which allowed the determination of sd values shown here. As a loading control, blots were stained with methylene blue prior to hybridization; this is shown below the autoradiograms. A to D, Probes for tRNAs. E to G, Probes for mRNAs. [See online article for color version of this figure.]

Figure 4.

Association of MatK with its target RNAs changes across tobacco development. A, Dot-blot analysis of the RNA from pellet (P) and supernatant (S) fractions of MatK:HA immunoprecipitations from the stroma of 7- and 25-d-old tobacco seedlings. C+, Plants tagged at the C terminus of MatK; N+, plants tagged at the N terminus of MatK. B, The ratios of pellet and supernatant signals were calculated and summarized for experiments with N+ and C+ plants. Means of these experiments are represented in a pie chart as ratios of the sum of all MatK targets. C, The ratio to ratio (25 d/7 d) is indicative of the developmental dependency of RNA-MatK interactions. This chart was derived from the experiment shown in A with immunoprecipitations from C- and N-terminally tagged plants considered as replicate experiments (error bars show sd). RNAs with values below 1 show a decrease in the association with MatK:HA in 25-d-old plants when compared with 7-d-old plants; values above 1 are the opposite.

Figure 5.

Model 1 with autoregulation of MatK reproduces the observed expression dynamics. Two possible models of matK gene expression were analyzed with respect to how well both models fit to our experimental data. A, Reaction scheme of the tRNA-MatK splicing network for model 1. The MatK transtargets trnA, trnV, and trnI are lumped into one tRNA species assigned as tRNA_sum. The values of trnA accumulation (Fig. 3A) were used to describe the expression dynamics of tRNA_sum, and tRNA_sum is assumed to be 10-fold more abundant than trnK (for details, see “Results”). k_1r and k_5r are the transcription rates of the tRNA precursors pre-trnK-matK (y1) and pre-tRNA_sum (y5), respectively. The protein MatK (y4) is encoded within the intron of the trnK-matK precursor gene and translated with the rate k_4p. As the levels of the tRNA precursors y1 and y5 as well as the MatK protein y4 increase, they form pre-trnK/MatK (y2) and pre-tRNA_sum/MatK complexes (y6), respectively. The pre-trnK/MatK repression complex (y2) inhibits the translation of matK via a negative feedback loop (red line with a blunt end). The subscript τ in k_2aτ and k_6aτ is the delay introduced in the models to signify the time taken for the formation of pre-trnK/MatK and pre-tRNA_sum/MatK complexes at day 25 of tobacco development. Within the tRNA/protein complexes, MatK splices the introns with rates k_3s and k_7s, leading to spliced tRNAs: mature trnK (y3) and mature tRNA_sum (y7). Competition of pre-trnK-matK (y1) and tRNA_sum (y5) for MatK (y4) is assigned by the factors and . To simplify the model, the involvement of nucleus-encoded splicing factors was not considered. Complex dissociation was assumed to be slow and, therefore, was neglected. Dashed arrows represent the degradation of RNAs, complexes, and the MatK protein with rates k_1d to k_7d. B, Accumulation of tRNA precursors, mature tRNAs, MatK protein, and tRNA/MatK complexes during tobacco development, derived from theoretical predictions of model 1 in comparison with northern-blot and western-blot analyses (E). Model 1 (top) fits certain experimental data displayed in E, such as the inverse correlation of MatK expression and matK mRNA (pre-matK-trnK) accumulation around day 7, the sharp decline of the MatK protein level after day 7, and the enrichment of pre-trnK/MatK complexes in mature seedlings. The model consistently reproduces the ratio-to-ratio value between pre-trnK/MatK complexes at days 25 and 7 (RR_25d/7d = 1.24), indicated with black arrows. Model 1 (bottom) shows temporal expression profiles of tRNA_sum (trnA, trnV, and trnI). The days of maximal accumulation of tRNA_sum are the same as those for trnK and those experimentally observed. C, Model 2 shows the same reaction scheme depicted in A, but negative autoregulation is removed from the system. D, Accumulation of tRNA precursors, mature tRNAs, MatK protein, and tRNA/MatK complexes during tobacco development derived from theoretical predictions of model 2 (C) in comparison with northern-blot and western-blot analyses (E). Model 2 (without autoregulation) fails to capture the distinctive expression dynamics of matK mRNA (pre-trnK-matK) and MatK protein. E, Experimental data for the accumulation of tRNA precursors, mature tRNAs, and MatK protein during tobacco development derived from northern-blot and western-blot analyses. The accumulation of MatK is plotted relative to the maximum obtained 7 dpi, while the abundance of the RNA precursor (pre-trnK-matK) and mature trnK is plotted relative to the maximum 25 and 59 dpi, respectively (set to 1). Reference parameters of the reaction kinetics of model 1 and model 2 are given in “Materials and Methods.” Note the different scaling.