The 5′ Guanosine Tracts of Human Telomerase RNA Are Recognized by the G-Quadruplex Binding Domain of the RNA Helicase DHX36 and Function To Increase RNA Accumulation

Abstract

Telomerase promotes telomere maintenance by copying a template within its integral RNA subunit to elongate chromosome ends with new telomeric repeats. Motifs have been defined within the telomerase RNA that contribute to mature RNA accumulation, holoenzyme catalytic activity, or enzyme recruitment to telomeres. Here, we describe a motif of human telomerase RNA (hTR), not previously characterized in a cellular context, comprised of several guanosine tracts near the RNA 5 end. These guanosine tracts together are recognized by the DEXH box RNA helicase DHX36. The helicase domain of DHX36 does not mediate hTR binding; instead, hTR interacts with the N-terminal accessory domain of DHX36 known to bind specifically to the parallel-strand G-quadruplex substrates resolved by the helicase domain. The steady-state level of DHX36-hTR interaction is low, but hTR guanosine tract substitutions substantially reduce mature hTR accumulation and thereby reduce telomere maintenance. These findings suggest that G-quadruplex formation in the hTR precursor improves the escape of immature RNP from degradation, but subsequently the G-quadruplex may be resolved in favor of a longer terminal stem. We conclude that G-quadruplex formation within hTR can stimulate telomerase-mediated telomere maintenance.Telomerase is an RNP reverse transcriptase that extends the ends of eukaryotic chromosomes by new telomeric repeat synthesis (2, 3). Enzyme activity requires the two universally conserved subunits of a functional telomerase RNP: the telomerase RNA and telomerase reverse transcriptase protein (TERT). Other proteins associate with telomerase RNA and/or TERT to promote their cellular accumulation and association or to engage the biologically active telomerase holoenzyme with telomere substrates (9, 34). In multicellular eukaryotes, somatic cells downregulate telomerase-mediated telomere maintenance as a tumor suppression mechanism (33). The progressive telomere attrition evident in most human somatic cell lineages with cell division cumulatively increases the likelihood of telomere unmasking as a signal for the DNA damage response (29). Telomerase activation is critical for long-term cellular proliferation, as reflected in the near-universal increase of telomerase subunit expression and activity in immortalized cell lines and cancers (20).Phylogenetic sequence comparisons, directed mutagenesis, and studies of disease-linked mutations have uncovered a complexity of sequence requirements for human telomerase RNA (hTR) folding, processing, and protein interactions (5, 10, 31). As a nascent transcript of RNA polymerase II, the hTR precursor recruits two sets of the H/ACA motif RNA binding proteins dyskerin, NHP2, and NOP10 in a chaperoned multistep process culminating in the exchange of RNP biogenesis factors for the fourth stably associated H/ACA motif RNP protein, GAR1 (13, 18). Proper assembly of the hTR H/ACA motif with dyskerin, NHP2, and NOP10 is essential for precursor maturation and produces the biologically stable telomerase RNP (10). The ...

Binding of G-quadruplexes to the N-terminal Recognition Domain of the RNA Helicase Associated with AU-rich Element (RHAU)

“…We have previously demonstrated that RHAU associates with a G-quadruplex at the 5Ј-end of human telomerase RNA (hTR) and is capable of unwinding this quadruplex in vitro (22). Interestingly, RHAU can associate with hTR in the context of the telomerase holoenzyme, an interaction that correlates with telomerase activity (23,24). G-tract substitutions in the quadruplex-forming region of hTR appear to confirm these results (23)(24)(25).…”

“…Interestingly, RHAU can associate with hTR in the context of the telomerase holoenzyme, an interaction that correlates with telomerase activity (23,24). G-tract substitutions in the quadruplex-forming region of hTR appear to confirm these results (23)(24)(25). Taken together, recognition and remodeling of quadruplex structures by RHAU play a central role in a number of key cellular regulatory processes.…”

Biogenesis of telomerase ribonucleoproteins

“…The 59 end of hTR contains several guanosine (G) tracts that increase mature RNA accumulation, likely by folding as a G-quadruplex that is subsequently resolved by the helicase DHX36 (Lattmann et al 2011;Sexton and Collins 2011). This region is missing from rodent TERs, consistent with the observation that G-quadruplex formation is stimulatory but not essential for hTR biogenesis (Sexton and Collins 2011). The 39 half of vertebrate TERs adopts a fold shared with the H/ACA family of RNAs (Fig.…”

Structure and function of echinoderm telomerase RNA

“…In addition to the template/pseudoknot and CR4/5 domains, there are immense species-specific structural differences within TR that appear in concert with the plethora of species-specific TR binding proteins. This includes RNA helicase RHAU, TCAB1, and box H/ACA dyskerin protein complex in vertebrates; Est1, Est3, Ku70/80 heterodimer, and Sm ring complex in fungi; p50 and p65 in ciliates; and box C/D Nop58 and MTAP (TCAB1 homolog) in flagellates (Mitchell et al 1999;Seto et al 1999;Hughes et al 2000;Venteicher et al 2009;Lattmann et al 2011;Sexton and Collins 2011;Egan and Collins 2012;Singh et al 2012;Tang et al 2012;Gupta et al 2013). These unique sets of TR accessory proteins are essential for TR localization, maturation, and RNP assembly (Schmidt and Cech 2015).…”