Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

<div><p>The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in <i>Arabidopsis</i> leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of <i>stn1</i> and <i>pol α</i> mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in <i>stn1</i> mutants may represent a manifestation of aberrant replication through telomeres.</p></div

Telomere structure in DNA polymerase α mutants.

<p>(A) TRF analysis. (B,C) Quantification of G-overhang signal in <i>pol α</i> (B) and <i>icu2-1</i> (C) mutants. The signals were normalized to the respective wild-type controls. Error bars represent SDs from three (wt-C24, <i>pol α</i>, wt-En-2) or four (<i>icu2-1</i>) independent samples. (D) Detection of blunt-ended telomeres by the hairpin ligation assay <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004682#pgen.1004682-Kazda1" target="_blank">[31]</a>. Ligation of a blunt-ended hairpin to chromosome ends covalently links the complementary telomeric DNA strands. Such cross-linked strands were separated from bulk of TRFs by alkaline electrophoresis and detected by Southern hybridization with the (TTTAGGG)₄ probe. Control reactions without the hairpin and in which the hairpin was cleaved by <i>Bam</i>HI are shown. The arrow-heads indicate signal from the blunt-ended telomeres. Asterisks indicates signal from intrachromosomal telomeric DNA. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from four biological replicates.</p

STN1 acts in the C-strand fill-in synthesis after G-strand extension by telomerase.

<p>(A) Frequency of phenotypic categories in G1 <i>stn1 tert</i> and G1 <i>exo1 stn1 tert</i> populations. (B) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. Average telomere length for each sample calculated by TeloTool is shown below the autoradiogram (C,D) Quantification of G-overhang signals from native gels (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004682#pgen.1004682.s003" target="_blank">Figure S3</a>). Errors represent SDs from three independent samples (two samples in the case of <i>exo1 stn1 tert</i>). Significance difference is indicated (two-tailed Student's test).</p

Impaired function of DNA polymerase α results in telomere deprotection.

<p>(A) Frequency of anaphases with bridges in floral tissues. (B,C) Amplification of chromosome end-to-end fusions by PCR in <i>pol α</i> and <i>icu2-1</i> mutants. Two combinations of subtelomeric primers specific for different chromosome arms (3L+1R and 2R+1L) were used. PCR products were detected by Southern hybridization with a telomeric probe. (D) Increased level of t-circles in <i>pol α</i>and <i>icu2-1</i> mutants measured by TCA. Signals from t-circles (arrowhead), TRFs, and interstitial telomeric DNA (asterisks) are indicated.</p

Effect of EXO1 on the structure of STN1-depleted telomeres.

<p>(A) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. (B) Quantification of the telomeric C-strand by dot-blot hybridization in G1 <i>stn1</i> and G1 <i>exo1 stn1</i> plants. Error bars show SDs from two independent samples; the P-value was calculated using a Student's t-test. (C) G-overhang analysis by the <i>in gel</i> hybridization technique. DNA samples pretreated with T4 DNA polymerase to remove 3′G-overhangs are indicated (3′ exo). The gels were first hybridized under nondenaturing conditions (top panels) and then denatured and hybridized again (bottom panels). (D) Quantification of G-overhang signals from a native gel. Error bars represent SDs from three (wt) and four (<i>stn1</i>; <i>stn1 exo1</i>) independent samples. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from five (wild-type) or four (<i>stn1</i>, <i>exo stn1</i>) biological replicates. Wild-type data are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004682#pgen.1004682-Kazda1" target="_blank">[31]</a>.</p

EXO1 aggravates the defects associated with STN1 depletion.

<p>(A) Phenotypic categories of <i>Arabidopsis stn1</i> mutants organized according to the severity of the observed growth defects. (B) Crossing scheme used to generate <i>exo1 stn1</i> mutants and control lines. Wild type and mutant alleles are indicated by capital and small letters, respectively (A – <i>EXO1A</i>, B – <i>EXO1B</i>, S – <i>STN1</i>). (C) Frequency of phenotypic categories in <i>stn1</i> and <i>exo1 stn1</i> mutant populations. The G2 populations were derived from the healthiest plants of the G1 generations. Number of plants scored is indicated in parentheses. (D) Frequency of anaphases with bridges in floral tissues. Number of plants scored is indicated in parentheses; at least 200 hundred anaphases were scored for each plant. The reduction of anaphase bridges in <i>stn1 exo1</i> compared to <i>stn1</i> mutants is highly significant (p<0.0001; two-tailed Student's t-test). (E) Expression of DDR genes in floral organs measured by qRT-PCR. Error bars represent SDs from three biological replicates; the asterisks denote a statistically significant difference to the <i>exo1 stn1</i> samples (Student's t-test; *p<0.05; **p<0.001; ***p<0.0001).</p

STN1 facilitates re-replication of heterochromatic regions.

<p>(A) Representative flow cytograms of the fluorescence intensity of nuclei extracted from the 5^th and 6^th true leaves of 4 weeks old plants. At least 10.000 nuclei were analyzed per sample. (B) qRT-PCR analysis of <i>PARP1</i> expression in two-week old seedlings grown on agar plates with or without Bleomycin (50 ng/mL). Error bars represent SDs from three biological replicates. (C) Representative flow cytograms of nuclei isolated from two week old seedlings; 3000 nuclei from several pooled seedlings were analyzed in each sample. Average X² and its SD obtained by testing the normality of the distribution of 8C nuclei in N samples is shown. X²<0.04 indicates that the peak has a normal distribution at the 95% confidence level. P values indicate significance of the difference of the X² statistic from <i>atrx5 atrx6</i>. (D) The scaled log2 ratios of genomic DNA Illumina reads from mutant 8C to wild type 8C nuclei are plotted across the chromosomes. The <i>atrx5 atrx6</i> and <i>stn1 atrx5 atrx6</i> data are derived from sequencing two independent samples.</p

Regulation of plant gene expression by alternative splicing

Abstract AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed &apos;combinatorial&apos; control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)-PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT-PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process