The hTR BIO box functions in collaboration with consensus elements of the H/ACA motif.
The biological accumulation of hTR but not any other studied human H/ACA RNA depends on a sequence motif in the loop of the 3′ hairpin (). The 3′ half of this hTR loop contains the biogenesis-promoting box (the BIO box) important for mature RNA accumulation, while the 5′ half of the loop contains a CAB (Cajal body) box dispensable for RNA accumulation but required for mature RNP concentration in Cajal bodies (17
). Function of both the BIO box and the CAB box depends on the integrity of the adjacent stem, and disruption of this stem imposes autosomal dominant DC (44
). Previously assayed BIO box sequence substitutions that eliminated mature hTR accumulation either replaced the entire loop or severely disrupted loop structure by introducing additional base pairing (17
). In search of a minimal change that selectively disrupts BIO box function without altering the length of the 3′ hairpin stem, we assayed additional sequence variants for their impact on mature hTR accumulation.
Fig 1 The BIO box has sequence and positioning requirements for function. (A) Secondary structure of full-length hTR and primary sequence of the 3′ hairpin upper stem-loop. Open boxes within the H/ACA domain indicate the location of the H box and ACA (more ...)
We expressed hTR precursor using the Pol II expression construct U3-hTR-500, which contains the U3 C/D box snoRNA promoter, mature hTR sequence, and 500 bp of downstream sequence from the endogenous locus. This construct efficiently produces 3′ processed hTR that is incorporated into the biologically active telomerase holoenzyme (17
). Total RNA was harvested from transfected human 293T cells, resolved by denaturing polyacrylamide gel electrophoresis (PAGE), and probed by Northern blotting to detect the accumulation of hTR and an internal loading control (LC). As demonstrated previously, the disruption of CAB box function with the substitution G414C did not inhibit mature hTR accumulation, while deletion of the 3′ hairpin upper stem-loop or loop replacement with a structured tetraloop reduced hTR accumulation to a level undetectable over the background of endogenous hTR in cells transfected with empty vector (, lanes 1 to 3 and 8 to 9; note that mature hTR often migrates as a doublet due to partial folding). To reduce loop interaction with CAB box binding proteins for purposes of the RNP assembly assays described below, we subsequently included the G414C CAB box substitution in “wild-type” hTR unless indicated otherwise.
Vertebrate telomerase RNAs share absolute conservation of the final loop residue that is hTR U418 () and >85% conservation of the preceding guanosine that is hTR G417 (37
). The nuclear magnetic resonance (NMR) structure of a model hTR 3′ hairpin stem-loop suggests that G417 pairs with U411 (41
). Although the G417C substitution strongly reduced hTR RNP accumulation monitored by RNase protection (41
), replacement of G417 with adenosine had little impact (, lane 4), perhaps because A-U base pairing would still be possible. In contrast, replacement of the absolutely conserved U418 with cytidine strongly reduced mature hTR accumulation (, lane 5). The combination of these two substitutions was no more deleterious than the U418C substitution alone (, lane 6). Replacing the entire 3′ hairpin loop with the U17 snoRNA 3′ hairpin loop CUGUC, which contains the BIO box sequence with an extra 3′ cytidine, did not provide full BIO box function (, lane 7). Because the U418C substitution substantially reduced mature hTR accumulation in vivo
without changing 3′ hairpin stem or loop length, we used this substitution for BIO box disruption in subsequent studies.
Surprisingly, it has not been addressed whether the BIO box functions autonomously or in coordination with the H/ACA motif. To investigate potential positioning-dependent coordination between the BIO box and the H/ACA motif, we altered or extended 3′ hairpin base pairing in the upper stem (). We found that deleting or pairing a predicted internal loop in the upper stem did not severely inhibit mature hTR accumulation (, lanes 3 to 5). In contrast, insertion of additional base pairs in the upper stem reduced mature hTR accumulation in proportion to the increase in stem length: insertion of two base pairs only partially inhibited hTR accumulation, while the insertion of eight base pairs reduced hTR accumulation to a level undetectable above background (, lanes 6 to 7). Similar inhibition of mature hTR accumulation was observed when five or eight base pairs were added to a 3′ hairpin upper stem that retained the wild-type internal loop (, lanes 8 and 9). Accumulation was not restored by insertion of 11 base pairs, which would present the BIO box on the same helical face as in wild-type hTR (, lane 10). We also attempted to functionally transplant an ectopic BIO box to other positions of mature or precursor hTR without achieving a rescue of the accumulation defect imposed by U418C substitution of the native BIO box (data not shown). Together, these results suggest that the physical spacing between the BIO box and the 3′ hairpin pocket is critical for BIO box function.
BIO box function does not require Pol II-coupled precursor expression or 3′ end formation.
The BIO box remains critical for RNA accumulation even if the noncanonical hTR H/ACA motif is swapped for a canonical H/ACA snoRNA (17
). However, BIO box function is not required if the hTR H/ACA domain accumulates by processing from a spliced intron rather than an unspliced autonomous transcript (41
). Together, these findings suggested that BIO box function is coupled to transcription context. This hypothesis has support from precedent because transcription-coupled association of processing factors with nascent yeast H/ACA snoRNA transcripts determines their specificity of 3′ end formation and processing as snoRNAs (40
To investigate whether BIO box function is dependent on hTR H/ACA domain expression as an autonomous transcript of Pol II, we compared the BIO box dependence of hTR H/ACA domain accumulation in a Pol II versus Pol III expression context (). Pol III does not support the synthesis of full-length hTR due to the presence of internal polyuridine tracts (32
). Therefore, the U3-hTR H/ACA-500 expression vector, which includes only the hTR H/ACA domain (nt 203 to 451), was minimally altered to create U6-hTR H/ACA-500, which replaces the human U3 C/D-box snoRNA Pol II promoter with the human U6 small nuclear RNA Pol III promoter. The hTR H/ACA domain has been shown to be 5′ and 3′processed from a full-length hTR transcript in vivo
). As expected from previous work, BIO box mutation reduced hTR H/ACA domain accumulation from the Pol II-transcribed precursor (, lanes 1 to 3). Unexpectedly, BIO box mutation also reduced the accumulation of a Pol III-transcribed RNA that migrates with the mobility expected for a properly 5′- and 3′-processed hTR H/ACA domain (, lanes 4 and 5). Based on blot hybridization results (data not shown), longer RNAs produced by Pol III are likely to be polyuridine-terminated primary transcripts protected from 3′-end trimming by La protein. We conclude that although the efficiency of processing is affected by transcription context, the BIO box can promote hTR H/ACA RNP accumulation independent of the transcribing polymerase and its associated factors.
Fig 2 The BIO box enhances RNP accumulation in different transcription contexts. (A and B) Construct schematics and results for BIO box function in Pol II and Pol III expression contexts. The hTR H/ACA domain (nt 203 to 451) with a wild-type (WT) 3′ (more ...)
Even if forced to be uncoupled from primary transcript synthesis, the BIO box could still function to recruit productive 3′-end processing machinery. For example, the BIO box could promote endonucleolytic cleavage of the primary transcript to override polyadenylation-induced nuclear export or degradation. This model has the appeal of accounting for a complete lack of a recognizable or required 3′ end formation signal downstream of mature hTR at its genomic locus (15
). We tested this model by inserting a self-cleaving ribozyme (RZ) after the 3′ end of mature hTR in an attempt to rescue the accumulation defect imposed by BIO box mutation (). We inserted either a minimal hammerhead RZ or a hepatitis delta virus RZ, the latter of which is reported to undergo particularly rapid cotranscriptional cleavage (16
). Surprisingly, although either RZ improved the accumulation of wild-type or BIO box mutant hTR, neither rescued the relative accumulation deficit imposed by BIO box disruption (, compare lane 2 to lanes 3 and 4 and lane 5 to lanes 6 and 7 as well as lanes 2 to 4 to lanes 5 to 7). These findings suggest that the formation or persistence of a precursor 3′ end can be rate limiting for hTR accumulation, such that RZ self-cleavage and/or formation of a nuclease-resistant 2′3′-cyclic phosphate promotes mature hTR production. In addition, toward understanding the role of the BIO box, the findings above indicate that the hTR functional requirement for a BIO box is independent of the precursor 3′ end formation mechanism.
The BIO box promotes hTR 3′ hairpin RNP assembly.
Instead of acting at an hTR-specific step of RNA processing, the BIO box could act at an early RNP assembly step by directly binding to a recruiting factor for H/ACA proteins. In this scenario, the hTR-specific requirement for the BIO box would derive from the need for hTR RNP assembly to compete with the more rapid degradation of its precursor relative to intron-encoded H/ACA RNA precursors, which would be protected from exonucleolytic degradation within a host mRNA transcript. Consistent with the hypothesis above, RNP assembly monitored by electrophoretic mobility shift assay (EMSA) indicated that HeLa cell extract assembled a distinct complex on the hTR H/ACA domain in addition to the complexes formed by both hTR and H/ACA snoRNAs (11
). The hTR 3′ hairpin with a wild-type CAB box and BIO box (both uncharacterized at the time) could compete for RNP assembly on the entire hTR H/ACA domain (11
). We therefore used HeLa whole-cell extract to determine whether the BIO box has a stimulatory role in RNP assembly on a pretranscribed RNA. We used radiolabeled hTR 3′ hairpin with the internal loop of the upper stem paired to promote stable folding (variant B in ) and with the CAB box disruption G414C to inhibit loop binding by the proteins that mediate Cajal body localization. RNA containing only these substitutions was the wild-type backbone for BIO box disruption by U418C substitution or ACA disruption by trinucleotide replacement with UGU (BIO and ACA variants, respectively).
When incubated with HeLa whole-cell extract, the reference wild-type hTR 3′ hairpin assembled an RNP with a discrete shift in electrophoretic mobility (, lanes 1 to 2). This RNP assembly was sensitive to increasing salt concentration but was still detectable in the presence of 300 mM NaCl (, lanes 2 to 4). RNPs assembled by the BIO and ACA variant RNAs migrated faster than RNPs assembled on the wild-type RNA and were nearly eliminated by only 200 mM NaCl (, lanes 6 to 8 and 10 to 12). Disruption of the salt-stable RNA mobility shift by BIO box or ACA mutation suggested that the BIO box could have a role in promoting RNP assembly.
Fig 3 The BIO box stimulates RNP assembly on the hTR 3′ hairpin. Radiolabeled hTR 3′ hairpin probes were added to HeLa whole-cell extract with RNP assembly monitored by EMSA. In panel A, wild-type (WT) RNA corresponds to nt 381 to 451 of hTR (more ...)
To better compare the relative RNP assembly efficiencies of the wild-type versus BIO and ACA variant RNAs, we measured the ability of unlabeled competitor RNAs to inhibit RNP assembly on the radiolabeled wild-type RNA (). Various concentrations of unlabeled competitor RNA were preincubated with whole-cell extract and other buffer components before radiolabeled wild-type 3′ hairpin RNA was added. Unlabeled wild-type RNA inhibited RNP assembly to half-maximum at a concentration of 0.1 ng/μl, an approximately 20-fold excess over the limiting radiolabeled RNA (, lane 4). In contrast, BIO box mutant RNA at over 2,000-fold excess or ACA mutant RNA at over 20,000-fold excess was required to achieve half-maximal inhibition (, lane 9 or 13). We conclude that the U418C BIO box mutation reduced hTR 3′ hairpin RNP assembly efficiency significantly but to a lesser extent than mutation of the ACA motif. This parallels the impact of BIO box or ACA mutation on hTR accumulation in vivo.
The BIO box promotes direct H/ACA protein-RNA interaction.
The BIO box and ACA dependence of RNP assembly on a pretranscribed RNA suggested that whole-cell extract reconstituted a physiological H/ACA RNP complex, but this was not conclusively established by EMSA alone. To further investigate the nature of the RNP assembled in whole-cell extract, we used extracts from HeLa cells overexpressing a protein of interest N-terminally tagged by fusion to tandem IgG binding domains (the ZZ tag). All tagged proteins used here were robustly and comparably overexpressed in transfected cells, as quantified by immunoblotting of whole-cell extract (data not shown). After radiolabeled RNA incubation in cell extract, RNPs containing the tagged protein were recovered on IgG resin, washed, and quantified using denaturing PAGE. In this assay, the amount of recovered radiolabeled RNA indicates the success of its assembly into RNP complexes containing the protein of interest.
We first applied this approach to the H/ACA RNP proteins dyskerin, NHP2, and GAR1 (NOP10 is refractory to tagging, but since it is the bridge between dyskerin and NHP2, its presence can be inferred). To reduce extract-mediated degradation of the radiolabeled RNA during affinity purification, we tagged the hTR 3′ hairpin at its 5′ end with the Pseudomonas
phage 7 (PP7) stem-loop binding site for coat protein (22
), which we found to stabilize the recombinant RNA in extract even without added PP7 coat protein. Dyskerin, NHP2, and GAR1 all assembled on the hTR 3′ hairpin, with an efficiency reduced by BIO box mutation and abrogated entirely by ACA mutation (). Cell extract containing no tagged protein was used as a specificity control (, lanes 13 to 15). Similar results were obtained using untagged hTR 3′ hairpin RNAs (data not shown). We also verified that this specificity was not due to enhanced degradation of the BIO and ACA variant RNAs in extract, as judged by equal recovery of radiolabeled RNAs from the unbound extract fraction following RNP affinity purification (data not shown). We conclude that the BIO box strongly stimulates H/ACA RNP assembly on the hTR 3′ hairpin in whole-cell extract.
Fig 4 RNP assembly specificity is recapitulated with a purified protein scaffold. (A and C) HeLa whole-cell extracts from cells transfected to express the indicated ZZ-tagged protein or untransfected cells (Mock) were incubated with radiolabeled PP7-tagged (more ...)
We then modified the reconstitution assay, reversing the immunopurification and RNA incubation steps. The tagged protein alone and any complexes containing the tagged protein were isolated by binding to IgG resin, washed resin was incubated with radiolabeled RNA, and, after removal of free RNA with additional washes, any RNP-assembled RNA was recovered and resolved by denaturing PAGE. In this assay, complexes containing tagged dyskerin or NHP2 but not GAR1 could assemble on the hTR 3′ hairpin, and this assembly was reduced by BIO box mutation and eliminated by ACA mutation (). Because GAR1 joins an H/ACA RNP late in its maturation, our RNP assembly assay with prepurified complexes recapitulates the physiological specificity of H/ACA RNP assembly: GAR1 should not purify an RNA-free protein complex that can load recombinant RNA, but if RNA is assembled into RNP in extract, additional maturation steps including the recruitment of GAR1 can occur.
We next examined the specificity of NAF1 interaction. Tagged NAF1 copurified the hTR 3′ hairpin RNA following RNP assembly in extract (, lanes 7 to 9), and prepurified protein complexes with tagged NAF1 bound RNA directly (, lanes 7 to 9). In either case, RNP formation was strongly reduced by BIO box mutation and eliminated by ACA mutation. Overall, the findings above indicate that NAF1, dyskerin, NHP2, and, by extension, NOP10 form a preassembled protein complex that is capable of binding the hTR 3′ hairpin. Also, at least under our extract RNP assembly conditions, the exchange of NAF1 for GAR1 remains incomplete. More surprisingly, BIO box-dependent hTR RNP reconstitution with purified, NAF1-bound core proteins suggests that the BIO box binding partner is part of this preassembled protein scaffold.
The preassembled core protein scaffold lacks other known chaperones.
Cellular biogenesis of H/ACA RNPs requires numerous factors, in addition to NAF1, that interact with dyskerin, NOP10, and/or NHP2 and assist in the RNA processing and RNP assembly steps preceding formation of a mature RNP. To evaluate whether these factors associate with RNP assembled in extract or, of special interest, form part of the preassembled NAF1/dyskerin/NOP10/NHP2 scaffold competent for direct RNA binding, we expressed tagged versions of known H/ACA RNP biogenesis factors and assayed their hTR interaction using both reconstitution protocols. Assembly of radiolabeled hTR 3′ hairpin in extract followed by RNP purification detected BIO box and ACA-dependent interaction of two more RNP assembly chaperones: NUFIP and RUVBL2 (, lanes 4 to 6 and 10 to 12). Similar assays of additional factors required for hTR biogenesis in vivo, including the nuclear cap methyltransferase sTGS1 (, lanes 13 to 15), failed to detect associations with the extract-assembled hTR 3′ hairpin RNP.
Notably, neither NUFIP nor RUVBL2 was part of the preformed protein scaffold competent for direct RNA binding (, lanes 4 to 6 and 10 to 12). Assays of additional factors required for hTR biogenesis in vivo, including sTGS1 (, lanes 13 to 15), also did not copurify the assembly-competent protein scaffold. Despite many years of investigation, it remains possible that some H/ACA RNP assembly factors remain to be discovered, but the most likely explanation to account for the results above is that a scaffold containing only NAF1, dyskerin, NOP10, and NHP2 is the platform for direct binding to an H/ACA RNA precursor. Other biogenesis factors that associate with an H/ACA RNP reconstituted in cell extract are not part of this assembly-competent scaffold prior to RNA loading. Furthermore, because hTR association with the preassembled protein scaffold remains dependent on the BIO box even when the scaffold is purified from extract prior to RNP assembly, the scaffold itself provides the BIO box-dependent RNP assembly stimulation.
No high-resolution structure of a eukaryotic core heterotrimer is available, with or without bound RNA. Of the core heterotrimer proteins, only NHP2 would be positioned suitably for direct contact with the BIO box (20
). Consistent with results from a previous study of yeast Nhp2p (21
), we find that recombinant human NHP2 alone binds the hTR 3′ hairpin without specificity for the BIO box (data not shown). A measurable sequence preference of NHP2 binding is likely to require its structural constraint by protein-protein interaction(s). We suggest that protein preassembly to form the NAF1/dyskerin/NOP10/NHP2 scaffold biases the conformational heterogeneity observed in recent solution structures of isolated yeast Nhp2p (26
) to favor the NHP2 conformation with a binding preference for the loop uridine presented by the hTR BIO box.
The preassembled core protein scaffold supports concerted two-hairpin RNP assembly.
Results above demonstrate that hTR H/ACA RNP assembly is dependent on the hTR BIO box in vitro
as well as in vivo
, but other human H/ACA RNAs lack this motif. To investigate the in vitro
RNP assembly properties of other human H/ACAs in comparison to hTR, we created a panel of one- or two-hairpin RNAs based on the validated secondary structures of the human U17, U64, and ACA28 snoRNAs (5
). U17 is the only human H/ACA RNA known to function in rRNA precursor cleavage rather than pseudouridylation, and it is the only known individually essential H/ACA RNA in yeast (24
). Like hTR, U17 has a noncanonical 5′ hairpin structure and a 3′ hairpin that can compete for full-length U17 RNP assembly in HeLa nuclear extract (11
We first assayed RNP assembly in extract for the radiolabeled two-hairpin U17, U64, and ACA28 snoRNAs and a comparably snoRNA-sized version of hTR (), which lacks the 5′ hairpin extension for telomerase catalytic activation (). We also assayed RNP assembly on the single 3′ hairpin of each snoRNA. The two-hairpin and 3′ hairpin hTR and U17 RNAs each assembled an H/ACA RNP in extract, as monitored by postassembly purification of tagged NHP2 (, lanes 8, 9, 11, 13, and 14). In contrast, although the two-hairpin versions of U64 and ACA28 assembled an RNP, the 3′ hairpin of either snoRNA alone did not (, lanes 26 to 29). Notably, the BIO box enhanced RNP assembly even for the snoRNA-like two-hairpin version of hTR (, compare lanes 9 and 10 and lanes 11 and 12). Parallel controls for background binding used cell extracts with no tagged protein (, mock purification lanes). The isolated 5′ hairpins from hTR, U17, or other snoRNAs failed to assemble an RNP (data not shown). The same results were obtained using tagged dyskerin instead of tagged NHP2 (data not shown).
Fig 5 Pseudouridylation guide RNAs show concerted two-hairpin RNP assembly. (A) HeLa whole-cell extracts from cells transfected to express ZZ-tagged NHP2 were incubated with radiolabeled 3′ hairpin (hp) or two-hairpin hTR (WT or BIO variant), U17, U64, (more ...)
We next assayed the panel of single-hairpin and two-hairpin RNAs by RNP reconstitution following protein purification. To our surprise, complexes containing tagged NHP2 () or tagged dyskerin (data not shown) that were purified prior to RNP assembly supported not only the same specificity of single-hairpin RNP assembly characterized in extract but also robust two-hairpin-dependent RNP assembly (, lanes 3 and 4). This finding suggests that the purified, resin-immobilized NAF1/dyskerin/NOP10/NHP2 scaffold achieves concerted assembly of core proteins on two H/ACA motif hairpins. Although we cannot exclude some potential for physical association of two separately immobilized NAF1-bound core protein complexes, the results above suggest that the preassembled scaffold for productive RNP assembly could already contain two sets of core proteins (). This scaffold architecture would account for why eukaryotic H/ACA RNAs all have two hairpins, whereas archaeal RNAs that assemble directly with dyskerin/NOP10 and separately with L7Ae do not. Unfortunately, we found the RNA-binding-competent NAF1/dyskerin/NOP10/NHP2 scaffold to be unstable to stringent washes and/or extended additional purification, hampering direct physical characterization of the functional scaffold architecture.
Distributed features of the hTR 3′ hairpin cooperate to promote RNP assembly.
The BIO box alone should contribute only a small fraction of the RNA binding surface for an H/ACA core protein complex, so we considered the possibility that it is necessary but not sufficient for stimulation of hTR 3′ hairpin RNP assembly. We constructed a series of chimeric 3′ hairpin RNAs that contained combinations of elements from hTR and ACA28 (). Chimeras 1 and 2 contained ACA28 sequence with the hTR 3′ loop and also pocket region sequences, including deletion of two base pairs above the pocket and insertion of two base pairs below the pocket to make the stem lengths more similar to those of hTR. In addition, in chimera 1 an adenosine was inserted in the 5′ side of the upper stem and a guanosine was inserted in the 3′ side of the lower stem to make internal loop and bulge sizes identical to hTR. Efficient RNA assembly with H/ACA proteins in extract was not supported by transplant of either the hTR loop or pocket sequences; instead, these chimeric RNAs showed only low, near-background purification similar to the ACA28 3′ hairpin (, lanes 8 to 11). We next transplanted the hTR 3′ hairpin loop and its atypically long lower stem into the ACA28 3′ hairpin backbone to generate chimera 3. The combination of these two hTR elements conferred a robust and reproducible improvement of RNP assembly (, lane 12) that was more than provided by the loop alone (lane 14) or the undetectable RNP assembly with transplant of the lower stem alone (lane 13). Nonetheless, even the combination of loop and lower stem did not fully recapitulate the RNP assembly efficiency of the intact hTR 3′ hairpin (, lane 8). Consistent with the findings above, partially unpairing the 3′ hairpin lower stem to produce a more typical stem length severely reduced mature hTR accumulation in vivo (data not shown). An atypically long lower stem could be precluded in modification guide RNAs by competing structural requirements for biological function (see Discussion). These results suggest that multiple features of the hTR 3′ hairpin act in a synergistic manner to increase RNA interaction affinity with the preassembled NAF1-bound core protein scaffold.
Fig 6 Several hTR 3′ hairpin features contribute to RNP assembly enhancement. HeLa whole-cell extracts from cells transfected to express ZZ-tagged NHP2 were incubated with a radiolabeled hTR 3′ hairpin, ACA28 3′ hairpin, or chimeric (more ...) Disease-associated H/ACA protein variants affect different steps of RNP assembly.
Finally, we investigated a potential connection between hTR sequence-mediated enhancement of H/ACA RNP assembly and the specificity of telomerase deficiency in DC. We overexpressed tagged disease-associated variants of dyskerin and NHP2 in HeLa cells and used the resulting whole-cell extract for reconstitution assays. All of the dyskerin variants supported hTR 3′ hairpin RNP assembly in both reconstitution assays ( and , lanes 2 to 9). This observation is consistent with the modest, if any, inhibition of RNA binding observed when dyskerin variants were assembled as core heterotrimers in reticulocyte lysate (43
). On the other hand, the NHP2 variants imposed severe defects in RNP assembly ( and , lanes 11 to 13). NHP2 variants expressed by transient transfection of 293T cells showed reduced association with hTR as well as other snoRNAs (), indicating that their assembly defect is not specific to hTR. These findings also parallel results from studies using reticulocyte lysate for RNP reconstitution (43
). Importantly, because NHP2 variants are expressed heterozygously in DC patient cells, they would decrease but not eliminate functional NHP2. In sum, for the overexpressed disease-linked dyskerin and NHP2 variants tested here, we did not find an RNP assembly defect specific to hTR. Instead, we suggest that in their physiological genomic contexts, DC mutations impose phenotypes by reducing the cellular level of the preassembled RNA-binding-competent H/ACA core protein scaffold. This could result in a more dramatic reduction of mature hTR than other H/ACA RNAs, due to a differentially sensitized balance of precursor RNP assembly versus degradation.
Fig 7 DC variants of NHP2 but not dyskerin inhibit hTR 3′ hairpin RNP assembly. (A) HeLa whole-cell extracts from cells transfected to express the indicated TAP-tagged dyskerin or ZZ-tagged NHP2 variant were incubated with radiolabeled PP7-tagged hTR (more ...)