Dragony Fu

Distinct Biogenesis Pathways for Human Telomerase RNA and H/ACA Small Nucleolar RNAs

Functional RNAs are processed according to cues from precursor expression context. The presence of an H/ACA motif within the human telomerase RNA (hTR) suggested that telomerase might utilize the biogenesis pathway of an H/ACA small nucleolar RNA. Here, we have investigated the requirements for processing, stability, and function of hTR. Instead of a biogenesis pathway dependent on transcription context or precursor sequence, we find that maturation of hTR requires cooperation of the H/ACA motif and an additional motif unique to the hTR H/ACA domain. This telomerase-specific hTR element is mutated in dyskeratosis congenita, and the disease-associated hTR substitution impairs RNA accumulation. Disease-associated hTR variants with sequence changes outside the H/ACA domain do not affect RNA processing or stability; they instead impose a catalytic defect. Our results reveal differences between the biogenesis of hTR and other H/ACA-motif RNAs and demonstrate distinct mechanisms of telomerase inhibition in human disease.

Human ALKBH7 is required for alkylation and oxidation-induced programmed necrosis

Programmed necrosis has emerged as a crucial modulator of cell death in response to several forms of cellular stress. In one form of programmed necrotic cell death, induced by cytotoxic alkylating agents, hyperactivation of poly-ADP-ribose polymerase (PARP) leads to cellular NAD and ATP depletion, mitochondrial dysfunction, reactive oxygen species formation, and ensuing cell death. Here, we show that the protein encoded by the human AlkB homolog 7 (ALKBH7) gene plays a pivotal role in DNA-damaging agent-induced programmed necrosis by triggering the collapse of mitochondrial membrane potential and large-scale loss of mitochondrial function that lead to energy depletion and cellular demise. Depletion of ALKBH7 suppresses necrotic cell death induced by numerous alkylating and oxidizing agents while having no effect on apoptotic cell death. Like wild-type cells, ALKBH7-depleted cells undergo PARP hyperactivation and NAD depletion after severe DNA damage but, unlike wild-type cells, exhibit rapid recovery of intracellular NAD and ATP levels. Consistent with the recovery of cellular bioenergetics, ALKBH7-depleted cells maintain their mitochondrial membrane potential, plasma membrane integrity, and viability. Our results uncover a novel role for a mammalian AlkB homolog in programmed necrosis, presenting a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.

Disease-Associated Human Telomerase RNA Variants Show Loss of Function for Telomere Synthesis without Dominant-Negative Interference

ABSTRACT Telomerase adds simple-sequence repeats to chromosome ends to offset the terminal sequence loss inherent in each cycle of genome replication. Inherited mutations in genes encoding subunits of the human telomerase holoenzyme give rise to disease phenotypes including hematopoietic failure and pulmonary fibrosis. Disease-associated variants of the human telomerase RNA are expressed in heterozygous combination with wild-type telomerase RNA. Here, we exploit a sensitized human primary cell assay system to investigate the biological function of disease-linked telomerase RNA variants and their impact on the function of coexpressed wild-type telomerase RNA. We find that telomerase RNA variants discovered in patients with dyskeratosis congenita or aplastic anemia show loss of function without any indication of dominant-negative impact on telomere maintenance by the coexpressed wild-type RNA. To reconcile this result with contradictory findings from reconstitution assays in vitro, we demonstrate that the lack of dominant-negative impact on telomere maintenance correlates with physiological assembly of active human telomerase holoenzyme ribonucleoproteins harboring monomers rather than higher-order multimers of telomerase RNA and telomerase reverse transcriptase. These findings support loss of function of telomerase RNA as a general mechanism of human disease.

Direct Repair of 3,N4-Ethenocytosine by the Human ALKBH2 Dioxygenase is blocked by the AAG/MPG Glycosylase

Exocyclic ethenobases are highly mutagenic DNA lesions strongly implicated in inflammation and vinyl chloride-induced carcinogenesis. While the alkyladenine DNA glycosylase, AAG (or MPG), binds the etheno lesions 1,N(6)-ethenoadenine (ɛA) and 3,N(4)-ethenocytosine (ɛC) with high affinity, only ɛA can be excised to initiate base excision repair. Here, we discover that the human AlkB homolog 2 (ALKBH2) dioxygenase enzyme catalyzes direct reversal of ɛC lesions in both double- and single-stranded DNA with comparable efficiency to canonical ALKBH2 substrates. Notably, we find that in vitro, the non-enzymatic binding of AAG to ɛC specifically blocks ALKBH2-catalyzed repair of ɛC but not that of methylated ALKBH2 substrates. These results identify human ALKBH2 as a repair enzyme for mutagenic ɛC lesions and highlight potential consequences for substrate-binding overlap between the base excision and direct reversal DNA repair pathways.