Translin and Trax differentially regulate telomere-associated transcript homeostasis

Abstract

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Keywords: C3PO; Chromosome Section; TERRA; Translin; Trax; telomeres.

Figure 1. Tfx1 (Trax), but not Tsn1 (Translin) regulates sub-telomeric transcript levels in S. pombe

Both images show relative transcript levels obtained from tiling microarrays. A. tlh1 gene transcripts are elevated in a tfx1Δ mutant, but not in tsn1Δ mutant. Elevation is maintained in a tfx1Δ tsn1Δ double mutant. Base pair (bp) designation represents the nucleotide annotation position for S. pombe chromosome 1 (tlh1 ORF is on the reverse strand, hence reverse strand profile is shown). B. A similar profile is seen for the other annotated sub-telomeric tlh gene, tlh2. Fold change values are given within the plots (* = P<0.05).

Figure 2. Centromeric transcript levels remain unaltered upon loss of Tsn1 or Tfx1

Centromeric transcript levels remains unaltered in either tfx1Δ or tsn1Δ mutants (middle two profiles) compared to the wild-type (top profile). The profile for an ago1Δ mutant is given as a control for a desilencing mutant (bottom profile). The approximate spread of heterochromatic and centromere core regions are given (bottom line). The profiles are for the forward (right) and reverse (left) strand of cen1. The S. pombe nucleotide coordinates shown for cen1 are chromosome 1: 3,754,000 – 3,790,000. Both strands for cen2 and cen3 show similar inactivation in tfx1Δ and tsn1Δ mutants and are given in Figure S1 and S2 respectively.

Figure 3. Loss of Tfx1 results in a telomere-defective phenotype, but telomere length is unaltered

A. Suppression of TBZ sensitivity of an ago1Δ mutant is a feature of telomere regulator proteins [38]. The tfx1Δ mutant, like the taz1Δ mutant [38] (Figure S3), partially suppresses the ago1Δ TBZ sensitivity, whereas a tsn1Δ mutation does not. B. Southern blot probed with a telomere-specific probe showing that both tfx1Δ and tsn1Δ mutants have telomeres with lengths similar to the wild-type. The taz1Δ mutant had highly elongated telomeres. The OtrtΔ strain has circular chromosomes without telomeres and serves as a control for the probe specificity. C. Quantification of telomere fragment migration ratios demonstrates no statistically significant difference between the mean telomere lengths of the tfx1Δ, tsn1Δ and wild-type strains. Mean migration values for at least four repeats were obtained. Pairwise Student's t-tests were conducted between all telomere containing strains (i.e. not the Otrt strain) and the wild-type. All P values were > 0.05 with the exception of the wild-type vs. taz1⁺ analyses which was < 0.01. Error bars are standard deviations.

Figure 4. Translin and Trax regulate telomere-associated transcripts in fission yeast cells

A. A schematic of an S. pombe telomere showing the telomere (purple) and the sub-telomeric region (light blue; previously referred to as STE) [42]. The ARRET and TERRA transcripts are aligned to their approximate template location. Arrows indicate primer positions used for first strand cDNA synthesis and RT-PCR (for TERRAs cDNA priming used oC, PCR was primed using o2/o4; for ARRETs cDNA priming used o3 and PCR was primed using o2/o4; primer sequences and designations are derived from Greenwood and Cooper [42]). B. Agarose gels showing analytical RT-PCR for TERRAs (first strand oC) and ARRETs (first strand o3). No primer controls indicate no first strand cDNA primers, demonstrating that PCR products are not due to endogenous first strand priming. RT-PCT for the act1⁺ gene transcripts are given as a positive control. C. RT-qPCR for TERRAs and ARRETs in tsn1Δ and tfx1Δ cells. Error bars represent standard deviation. Student's t-test pairwise comparison were carried out for mean values between the wild-type vs. mutants. In all cases P values were > 0.05 with the exceptions of wild-type vs. tsn1⁺ for TERRA levels (upper set) and wild-type vs. tfx1⁺ for ARRET levels (lower set) where P values were < 0.01.

Figure 5. Human Translin and Trax influence TERRA levels differentially for distinct telomeres

RT-qPCR for human TERRAs for the telomeres of the q arm of chromosome 10 (left) and the q arm of the sex chromosome (X/Y; right) in TSN and TSNAX depleted SW480 cells. NI RNA = non-interfering RNA. Error bars represent standard deviations. Student's t-test pairwise comparison were carried out for mean values between NI RNA treated cells and siRNA depleted cells. Only comparisons marked with * showed statistical significance (P < 0.05).

Figure 6. Model for the mechanism of telomere-associated transcript differential control by Tfx1 and Tsn1 in S. pombe

Tfx1 serves to repress sub-telomeric ARRETs and tlh transcripts (upper red full/broken lines), but stabilizes (upper green arrow) elevated levels of TERRAs seen in the tsn1Δ mutant. Tsn1 plays a reciprocal role, normally suppressing TERRA levels (lower red line), but stabilizing (lower green arrow) the elevated ARRET levels (but not the elevated tlh transcript levels) seen in the tfx1Δ mutant.