TET (Ten-Eleven-Translocation) proteins are Fe(II) and α-ketoglutarate-dependent dioxygenases1-3 that modify the methylation status of DNA by successively oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxycytosine1,3-5, potential intermediates in the active erasure of DNA methylation marks5,6. We show here that IDAX/ CXXC4, a player in the Wnt signaling pathway7 that has been implicated in malignant renal cell carcinoma8 and colonic villous adenoma9, functions as a negative regulator of TET2 protein expression. IDAX/ CXXC4 was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The Idax CXXC domain binds DNA sequences containing unmethylated CpGs, localises to promoters and CpG islands in genomic DNA, and interacts directly with the catalytic domain of Tet2. Unexpectedly, Idax expression resulted in caspase activation and Tet2 protein downregulation, in a manner that depended on DNA-binding through the Idax CXXC domain. Idax depletion prevented Tet2 downregulation in differentiating mouse embryonic stem (ES) cells, and shRNA against IDAX increased TET2 protein expression in the human monocytic cell line U937. Notably, we find that the expression and activity of TET3 are also regulated through its CXXC domain. Taken together, these results establish the separate and linked CXXC domains of TET2 and TET3 respectively as novel regulators of caspase activation and TET enzymatic activity.

Mutations in the PARN gene (encoding poly(A)-specific ribonuclease) cause telomere diseases including familial idiopathic pulmonary fibrosis (IPF) and dyskeratosis congenita1,2, but how PARN deficiency impairs telomere maintenance is unclear. Here, using somatic cells and induced pluripotent stem cells (iPSCs) from patients with dyskeratosis congenita with PARN mutations, we show that PARN is required for the 3′-end maturation of the telomerase RNA component (TERC). Patient-derived cells as well as immortalized cells in which PARN is disrupted show decreased levels of TERC. Deep sequencing of TERC RNA 3′ termini shows that PARN is required for removal of post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for degradation. Diminished TERC levels and the increased proportion of oligo(A) forms of TERC are normalized by restoring PARN, which is limiting for TERC maturation in cells. Our results demonstrate a new role for PARN in the biogenesis of TERC and provide a mechanism linking PARN mutations to telomere diseases.

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Mutations in 11 genes have been described in patients with dyskeratosis congenita (DC) and related telomere diseases, and account for ~60% of cases. Amongst these, loss-of-function mutations in the poly(A)-specific ribonuclease (PARN) have most recently been found in patients with DC and idiopathic pulmonary fibrosis. PARN has no known role in telomere biology. Why PARN deficiency should mimic telomere diseases remains unclear. Based on its 7-methylguanylate (m7 G) cap recognition and poly(A) deadenylase functions, PARN's primary role is considered to be in regulating mRNA metabolism. Recent studies implicate PARN in non-coding RNA biogenesis, including maturation of small nucleolar RNAs (snoRNAs) via the deadenylation of oligo-adenylated (oligo(A)) intermediates. In two DC patients with biallelic defects in the PARN gene, we found decreased levels of the telomerase RNA component (TERC). TERC is the essential non-coding RNA template and scaffold of the telomerase holoenzyme. TERC possesses an m7 G cap but unlike mRNAs does not have a long poly(A) tail; rather, TERC contains a box H/ACA motif as is found in some snoRNAs. We hypothesized that TERC is regulated by PARN in a manner similar to snoRNAs, given their shared 3′ box H/ACA architecture. Here, usingsomatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation and accumulation of TERC RNA. In PARN-mutant iPS cells and PARN-deficient cell lines, we find impaired telomerase activity and telomere maintenance. Deep sequencing of TERC 3' ends reveals that PARN is required for the removal of genomically-encoded extensions and post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for destruction. In keeping with this, TERC transcripts from PARN-mutant patient cells decay at an accelerated rate. The diminished TERC steady-state levels, increased oligo(A) forms of TERC, and defects in telomere maintenance in patient cells are rescued by ectopic expression of PARN. Global transcript analysis by RNA-Seq in patient cells and PARN-deficient cell lines compared to controls reveals no protein coding mRNAs that consistently manifest a fold-change exceeding the change in TERC levels. Our data indicate that PARN functions in TERC biogenesis via deadenylation of oligo-adenylated nascent transcripts, which promotes 3' end maturation and stability. Given these findings and the phenotype of patients with PARN mutations, we speculate that a major, non-redundant function of PARN in human cells is regulating the maturation of TERC and other non-coding RNAs, more so than mRNA metabolism. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to defective telomere maintenance in DC and related diseases. Disclosures No relevant conflicts of interest to declare.