TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe (2016)
Figure 1

Figure 1. Polyadenylated TERRA largely devoid of telomeric sequences accumulates upon telomere shortening.

A Schematic representation of the S. pombe telomeric transcriptome. Telomeric repeats are shown in black, subtelomeric sequences in grey. Oligonucleotides used for RT-PCR and for 3’ end RACE are indicated.

B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).

C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.

D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student’s t test).

E ChIP experiments with anti-RNA polymerase II (RPII) antibodies and extracts from ter1+ and ter1Δ cells grown for ~ 43 pds. Quantitative PCRs were performed using oligonucleotides oF1 + oR1 (subtel) or oligonucleotides amplifying a region from the actively transcribed act1+ locus. Values are expressed as fold increase over ter1+. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to ter1+; two-tailed Student’s t test). The western blot to the right shows no difference in RNPII levels between ter1+ and ter1Δ cells used in ChIP experiments. Ponceau S staining serves as loading control.

F 3’ RACE analysis of polyA+ TERRA in ter1Δ cells grown for ~ 43 pds. The sequence at the top is the genomic reference sequence (gen) taken from the pNSU70 plasmid (nucleotide positions are indicated). The sequences below derive from all positive clones isolated. Sequences boxed in blue, green and yellow correspond to the oligonucleotide used for RACE (o3’RACE), subtelomeric DNA and telomeric repeats, respectively. Genomic adenines corresponding to putative RNA cleavage sites (CS) are highlighted.

G Western blot analysis of cellular fractions from ter1+ and ter1Δ cells grown for ~ 43 pds. Total proteins (tot) and equivalents of soluble (sol) and insoluble (ins) proteins were loaded. Actin serves as a control for soluble proteins, H3K9ac for chromatin-bound proteins.

H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.