Telomeres are essential nucleoprotein structures that protect linear eukaryotic chromosome ends from detrimental repair processes as well as natural replication-associated shortening (Hayflick, 1965; Harley et al., 1990; Wellinger and Zakian, 2012; de Lange, 2018). In stem, germ, and most cancer cells, the enzyme telomerase maintains telomeric DNA at a functional length, which is key for the unlimited proliferation capacity of these cells (Shay, 2016; Roake and Artandi, 2020). Telomerase synthesizes telomeric repeats de novo using a reverse transcriptase activity and its internal RNA as a template (Greider and Blackburn, 1985; Greider and Blackburn, 1987). Unicellular eukaryotes such as yeasts express telomerase constitutively. Notably, the overall telomerase composition and aspects of telomerase dynamics are strikingly conserved between yeast and higher eukaryotes (Vasianovich and Wellinger, 2017). This makes yeast a great model organism to study telomerase behavior and for predicting this process in higher eukaryotes.

Telomerase is a complex ribonucleoprotein machine. The non-coding RNA, called TLC1 in Saccharomyces cerevisiae, is at the heart of the telomerase holoenzyme. It harbors a template for telomeric DNA synthesis and acts as a scaffold for telomerase-associated proteins (Wellinger and Zakian, 2012). The repertoire of the budding yeast telomerase proteins includes the Est2 catalytic subunit and several additional proteins such as Est1, Est3, Pop1, Pop6, Pop7, the Sm_7, and Yku70/Yku80 complexes, which all are required for telomerase function in vivo. Like most yeast snRNAs, the major form of TLC1 is a poly-A^- RNA Pol II transcript, which has a TMG cap at the 5’-end and associates with the ring-shaped heptameric Sm₇ complex at its 3’-end (Chapon et al., 1997; Seto et al., 1999). This Sm₇ complex is crucial for RNA maturation, including 5’-TMG capping and 3’-end processing, and is absolutely essential for the stability of both telomerase and snRNAs (Rymond, 1993; Roy et al., 1995; Seto et al., 1999; Coy et al., 2013; Shukla and Parker, 2014).

TLC1 is an extremely low abundance RNA. According to different estimates, only 10–30 mature TLC1 molecules are present in a haploid yeast cell, thus severely limiting the quantity of functional telomerase complexes (Mozdy and Cech, 2006; Gallardo et al., 2008; Bajon et al., 2015). As a result, the 64 chromosome ends present in a haploid cell after DNA replication must rely on a very limited number of telomerase RNPs for their maintenance. In fact, the available nuclear telomerase arsenal may be even lower, as at steady state, 30–40% of TLC1 molecules reside in the cytoplasm (Gallardo et al., 2008). However, the reason for the cytoplasmic localization of telomerase RNA is still unclear.

One hypothesis stipulates that the TLC1 RNA shuttles to the cytoplasm as a part of its biogenesis process (Ferrezuelo et al., 2002; Gallardo et al., 2008). According to this model, TLC1 gets exported to the cytoplasm to associate with some of its protein subunits, followed by re-import of the RNP to the nucleus. However, which telomerase subunits bind to TLC1 in the cytoplasm is unclear. Furthermore, the current view of telomerase biogenesis suggests that the 3’-stabilizing Sm₇ ring binds telomerase RNA very early in its life cycle, prior to RNA export to the cytoplasm (Gallardo et al., 2008). Consistently, the SmB, D1, and D3 subunits of the Sm₇ complex contain NLS sequences, suggesting that the ring can get imported into the nucleus on its own (Bordonné, 2000). In addition, TMG capping, which requires Sm₇, appears functional when TLC1 export is blocked (Gallardo et al., 2008). Finally, since the Sm₇ complex is crucial for the stability of telomerase RNA (Seto et al., 1999), it would be expected to associate with TLC1 at the earliest possible stages. However, in both yeast and mammals, snRNAs that belong to the Sm₇ class are exported to the cytoplasm to assemble with the Sm₇ complex (Fischer et al., 2011; Becker et al., 2019). Therefore, if TLC1 follows a path similar to snRNAs, newly synthesized telomerase RNA may temporarily shuttle to the cytoplasm to assemble with the Sm₇ proteins, which would contradict the above model. In addition, the cytoplasm may also be the final destination for TLC1 molecules, where old dysfunctional telomerase RNAs would be degraded. Therefore, the cytoplasmic TLC1 fraction may be a complex mix of both old degrading molecules as well as newly synthesized RNA transcripts that undergo assembly with protein subunits.

There is evidence that nucleo-cytoplasmic export of telomerase RNA is mediated by the Xpo1/Crm1 exportin and re-import is dependent on Mtr10 as well as Kap122 (Ferrezuelo et al., 2002; Gallardo et al., 2008). However, molecular details of TLC1 transport by these factors are missing, for example it is unclear at what stage and in which state TLC1 may shuttle to the cytoplasm or whether it gets exported permanently for degradation.

The Xpo1/Crm1 exportin belongs to the family of karyopherins which transport cargo RNA and proteins through nuclear pores using a RanGTP – RanGDP gradient (Köhler and Hurt, 2007). Many RNA species, such as snRNAs, rRNAs, and tRNAs depend on Xpo1 for their export. In contrast, transport of mRNA relies on the Mex67 export receptor (Segref et al., 1997; Santos-Rosa et al., 1998), which is structurally unrelated to karyopherins and does not depend on a RanGTP gradient (Köhler and Hurt, 2007). Inactivation of Mex67 leads to nuclear accumulation of mRNA (Segref et al., 1997; Santos-Rosa et al., 1998). In addition, in the absence of Mex67, poly-A⁺ RNA appears to be extremely unstable, which was attributed to untimely RNA decay due to the export block (Tudek et al., 2018). Notably, Mex67 is also somehow implicated in the cytoplasmic export of Xpo1-transported cargoes, such as snRNA, rRNA, and tRNA, raising a question of why these RNA species require two export receptors (Yao et al., 2007; Faza et al., 2012; Chatterjee et al., 2017; Becker et al., 2019). In fact, the precise function of Mex67 and the molecular details of its RNA export role are unknown.

In this study, we aimed to dissect the dynamic, life-cycle-dependent localization of telomerase RNA molecules via single molecule fluorescence in situ hybridization (smFISH) microscopy. For this purpose, we required a tool that would discriminate and selectively visualize new vs pre-existing matured telomerase RNA transcripts within the total TLC1 population. Fluorescent imaging of telomerase RNA molecules tagged with the MS2 repeats has been successfully used in the past (Gallardo et al., 2011; Cusanelli et al., 2013; Bajon et al., 2015). However, constitutive RNA tagging has its limitations: it captures the general RNA population at its steady state and therefore cannot reveal the dynamics of specific RNA subpopulations, such as new or old TLC1 molecules. In order to identify these fractions in the total TLC1 population and to reveal their specific behavior, we converted the constitutive TLC1-MS2 tagging system into an inducible one using a recombination-induced tag exchange approach (Verzijlbergen et al., 2010). In this system, controlled induction of TLC1 tagging with the MS2 repeats results in gradual appearance of new TLC1-MS2 molecules, which can be easily distinguished from the bulk untagged TLC1 population using an MS2-specific FISH probe. At the same time, untagged TLC1 molecules that lack the MS2-specific signal will represent the pre-existing TLC1 fraction which for simplicity will be referred to as old TLC1 molecules.

Using this inducible TLC1 tagging tool, we show that very rapidly after transcription, newly produced telomerase RNA molecules are temporarily exported to the cytoplasm, consistent with the shuttling model. Old TLC1 transcripts also tend to accumulate in the cytoplasm likely for degradation purposes. Therefore, the results show that the cytoplasmic TLC1 RNA is per se a heterogeneous population comprised of both new and old telomerase RNA transcripts. The data also show that Xpo1 and Kap122, previously shown to influence subcellular TLC1 localization, affect the shuttling of newly synthesized TLC1 molecules. Notably, cytoplasmic export of TLC1 is entirely dependent on Xpo1, whereas telomerase RNA re-import to the nucleus seems to involve not only Kap122 but also other less efficient import mechanisms. Strikingly, Mex67 plays an entirely unexpected role in the biogenesis of telomerase RNA. While cells without the Xpo1 exportin sequester all newly synthesized TLC1 in the nucleus, disruption of Mex67 leads to a drastically different phenotype, resulting in a complete loss of new TLC1 transcripts. This phenotype is recapitulated in a mex67-5 xpo1-1 double mutant and, remarkably, is suppressed by inactivation of the nuclear exosome subunit RRP6. Thus, Mex67 is crucial for stabilization of telomerase RNA in the nucleus immediately after transcription and prior to export – a function that may be required for other RNA species as well. Finally, we show that similar to snRNAs, telomerase RNA assembles with the Sm₇ complex in the cytoplasm. This association is essential for the stability of TLC1 RNA in the cytoplasm and its re-import to the nucleus. Cytoplasmic association of telomerase RNA with the Sm₇ complex implies that the downstream Sm₇-dependent events such as 5’- and 3’-end RNA processing occur after TLC1 returns to the nucleus. Therefore, in this study we challenge the current view of telomerase RNA biogenesis by adding Mex67 as a novel player essential for nuclear TLC1 stability right after transcription, and by localizing the Sm₇-binding step to the cytoplasm after the export of TLC1. These results underscore the general utility and validity of an inducible tagging system to probe dynamic localization details during the life cycle of any RNA.

As for any RNA species, the cellular pool of telomerase RNA encompasses a variety of molecules at different stages of biogenesis. Conventional imaging methods that rely on RNA-specific probes or tags visualize the total RNA pool and therefore yield information about the steady state RNA distribution. However, the diverse characteristics of various RNA sub-populations generally remain hidden in such assays. In this study, we overcame this limitation by combining fluorescent imaging with an inducible RNA tagging approach and applying it to the telomerase RNA TLC1. In this system, an inducible recombination event leads to a rearrangement of the TLC1 genomic locus and the expression of TLC1 molecules tagged with MS2 repeats (Figure 1). Using a probe specific for the MS2 tag, we can selectively identify and analyze newly synthesized telomerase RNA transcripts within a heterogeneous TLC1 population and track their specific movements by smFISH (Figure 2). In addition, the dynamic behavior of TLC1 transcripts that remain untagged after recombination is complete, provides clues about the old or matured TLC1 molecules.

At steady state, 30–40% of telomerase RNA molecules localize in the cytoplasm (Figures 2D, 0 h) (Gallardo et al., 2008). However, the nature of the cytoplasmic TLC1 molecules remained enigmatic. According to the nucleo-cytoplasmic shuttling model, telomerase RNA gets exported to the cytoplasm for biogenesis purposes (Ferrezuelo et al., 2002; Gallardo et al., 2008). It was possible that TLC1 also accumulated in the cytoplasm at the end of its life cycle. Using the inducible TLC1 tagging approach, we show that in fact both models apply. Accordingly, we observed a temporary accumulation of newly synthesized TLC1 transcripts in the cytoplasm immediately after recombination induction, followed by their gradual return to the nucleus and re-establishment of a steady state RNA distribution (Figure 2E). Thus, our results provide direct evidence for the nucleo-cytoplasmic TLC1 shuttling model and narrow down this process to an early post-transcriptional event. Moreover, we show that old untagged TLC1 RNA accumulates in the cytoplasm by the end of the time course experiment (Figures 2D, 16 h), indicating a possible cytoplasmic decay of TLC1 molecules at the end of their life cycle. It still needs to be determined what triggers the exit of old transcripts from the nucleus. Nevertheless, a significant number of old TLC1 molecules remained in the nucleus by the end of the time course (Figures 2D, 16 h). Hence, it is possible that telomerase RNA may also get degraded via a nuclear mechanism. In addition, we cannot exclude that TLC1 RNA undergoes additional rounds of shuttling at later stages of its life cycle. However, we favour the idea that untagged TLC1 foci that remain in the nucleus are the result of late recombination events that occurred in a subset of cells (Figure 1C–G). In this scenario, by the end of the time course, the majority of untagged TLC1 molecules are truly ‘old’ and localize in the cytoplasm for degradation, whereas a subset of untagged TLC1 is still functional and operates in the nucleus. Consistent with this idea, telomerase RNA has a very long-half life and can remain functional for much longer than 60 min, exceeding the length of one full cell cycle (Chapon et al., 1997; Larose et al., 2007).

The Xpo1 exportin and Kap122 importin were previously shown to affect the subcellular distribution of telomerase RNA (Gallardo et al., 2008). However, we aimed to understand whether these karyopherins are responsible for the nucleo-cytoplasmic shuttling of newly synthesized telomerase RNA transcripts (Figure 3A–C). Importantly, our results show that export of new telomerase RNA is entirely dependent on the Xpo1 exportin. Accordingly, in cells with dysfunctional Xpo1, newly transcribed TLC1 molecules accumulated exclusively in the nucleus, which would not be the case if an additional export receptor participated in telomerase RNA export (Figure 3A,B). On the other hand, Kap122 importin promotes the re-import of new TLC1 molecules to the nucleus, as following export in kap122Δ cells, most TLC1 molecules remained in the cytoplasm (Figure 3A,C). However, unlike TLC1 export which depends exclusively on Xpo1, nuclear re-import of new telomerase RNA molecules appears to be mediated by several importins. Accordingly, at later stages after the recombination initiation, the nuclear TLC1 fraction gradually increased in kap122Δ cells (Figure 3C). This alternative pathway could be dependent on Mtr10, which was previously shown to affect subcellular TLC1 localization (Ferrezuelo et al., 2002; Gallardo et al., 2008). Existence of a secondary Kap122-independent re-import mechanism for newly synthesized TLC1 transcripts is also supported by the fact that telomere maintenance is only mildly affected in the absence of Kap122 (Figure 2—figure supplement 1D). Interestingly, the maximal level of newly synthesized TLC1 RNA is decreased in kap122Δ cells (Figure 3D,E), suggesting that TLC1 that cannot re-enter the nucleus is degraded during the cytoplasmic phase. Therefore, a cooperation of several importins, including Kap122, facilitates a quick and efficient return of new TLC1 transcripts to the nucleus (Figure 6). Failure or delay in such re-import may lead to loss of telomerase RNA.

Figure 6

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There is evidence that the karyopherin-unrelated export receptor Mex67 may cooperate with the Xpo1 exportin in the cytoplasmic export of several RNA species, including snRNAs, rRNAs and tRNAs (Yao et al., 2007; Faza et al., 2012; Chatterjee et al., 2017; Becker et al., 2019). However, the molecular mechanism of this Mex67 function is not well understood. Here, we decided to test whether Mex67 affects the nucleo-cytoplasmic shuttling of new telomerase RNA transcripts. Strikingly, newly synthesized TLC1 molecules almost completely disappear upon inactivation of Mex67 (Figure 4A–D). Using thiolutin-induced transcription block and RNA Pol II ChIP experiments, we ruled out the possibility that dysfunctional Mex67 causes the defect in TLC1 transcription (Figure 4F; Figure 4—figure supplement 1E). Instead, inactivation of Mex67 results in a nearly complete loss of new telomerase RNA after transcription – a phenotype that can be fully suppressed by removal of the Rrp6 exosome component (Figure 4C,E; Figure 4—figure supplement 1F,G). The few TLC1 foci that remained detectable upon inactivation of Mex67 almost exclusively localized at the nuclear border (Figure 4C; Figure 4—figure supplement 1C), suggesting that in the absence of Mex67, new TLC1 transcripts quickly degrade in the nucleus and never make it to the cytoplasm. In addition, new TLC1 RNA was also unstable in a mex67-5 xpo1-1 double mutant grown at the restrictive temperature (Figure 4—figure supplement 2), similar to the phenotype observed in mex67-5 single mutants (Figure 4A–D). Altogether, these data show that Mex67 is essential for the nuclear stability of newly transcribed telomerase RNA prior to its Xpo1-mediated cytoplasmic export. On the other hand, old TLC1 molecules, which remained untagged during the experiment, were not destabilized by inactivation of Mex67 (Figure 4C, Figure 4—figure supplement 1B).

An earlier study reported that in mex67-5 cells grown at the restrictive temperature, telomerase RNA molecules are lost from the cytoplasm, which was interpreted as nuclear accumulation of TLC1 RNA (Wu et al., 2014). Our data here show that newly synthesized molecules largely contribute to the cytoplasmic TLC1 fraction and their disappearance in the absence of Mex67 indeed results in a decrease in the cytoplasmic TLC1 RNA. Notably, in this situation the nuclear TLC1 signal is composed of only matured or old RNA and there is no accumulation of new TLC1 molecules in the nucleus. Hence, the power of our inducible RNA tagging approach allows a much more targeted and detailed assessment of the dynamics of the complex TLC1 RNA population.

Similar to our results, depletion of Mex67 was shown to cause rapid nuclear degradation of all newly transcribed poly-A⁺ mRNAs (Tudek et al., 2018). This untimely RNA decay was attributed to the role of Mex67 as an mRNA export receptor. Mechanistically, it was suggested that this effect involves Nab2 that binds to poly-A⁺ tails and protects RNA transcripts from degradation (Hector et al., 2002; Schmid et al., 2015). The authors hypothesized that upon export block by Mex67 inactivation, Nab2 cannot be recycled from its substrate. Thus, accumulating poly-A⁺ RNA tails will sequester the nuclear Nab2 pool away from newly formed RNA, rendering them unstable (Tudek et al., 2018). It is important to note that the vast majority of TLC1 (95%) is generated directly via the Nrd1/Nab3 non-coding RNA termination pathway (Noël et al., 2012). So far, Nab2 has not been implicated in this pathway, but we cannot exclude the possibility that other maturation factors specific to the non-coding RNA termination pathway could play a role similar to Nab2. Hence, the model suggested by Tudek et al. could also explain premature degradation of new TLC1 transcripts upon inactivation of Mex67 observed in our study (Figure 4). However, very much distinct from the above model, our results argue that Mex67 does not act as a classical TLC1 export receptor and an export block cannot explain the degradation of new TLC1 molecules. First, we show that export of new telomerase RNA molecules to the cytoplasm is entirely dependent on Xpo1 (Figure 3A,B). If Mex67 operated in parallel with Xpo1 in TLC1 export, at least a fraction of new telomerase RNA molecules would be expected to eventually reach the cytoplasm in xpo1-1 cells grown at the restrictive temperature, which we did not observe (Figure 3A,B). Second and more importantly, although inactivation of Xpo1 completely blocks TLC1 in the nucleus, these molecules are perfectly stable and their number even increases beyond the level observed in wild type cells (Figure 3D,E). This means that at least for telomerase RNA, an export block per se does not lead to nuclear RNA decay. In other words, while in cells with dysfunctional Mex67 newly transcribed TLC1 RNA is completely lost, it is retained and accumulates in the nucleus in xpo1-1 mutants grown at the restrictive temperature (Figure 4A–D). This suggests that Mex67 acts upstream of Xpo1 and plays an unexpected role in stabilization of newly transcribed TLC1 molecules before their cytoplasmic export (Figure 6).

Hence, our results indicate a novel role of Mex67 in stabilization of telomerase RNA which to our knowledge is the first time that Mex67 is implicated in a function distinct from RNA export. Mex67 is known to bind at least to some of its cargoes co-transcriptionally (Dieppois et al., 2006; Wende et al., 2019). It has been proposed that such early association with its RNA substrates may assist in proper 3’-end RNA maturation and protection (Forrester et al., 1992; Hammell et al., 2002; Qu et al., 2009). Failure to execute this function may lead to elimination of faulty transcripts as was previously reported for other 3’-end processing mutants (Minvielle-Sebastia et al., 1991; Forrester et al., 1992; Hammell et al., 2002). There was some evidence that Mex67 may be able to associate with telomerase RNA (Wu et al., 2014), and our results here firmly establish that Mex67 protects the 3’-end of TLC1 from nuclear degradation by Rrp6 (Figure 4C,E; Figure 4—figure supplement 1F–H; Figure 6). Moreover, since Mex67 can bind to both RNA and nuclear pore components, it may be responsible for a quick delivery of new transcripts to the nuclear pore where they are stabilized and exported by Xpo1. The latter is consistent with our finding that the few newly synthesized molecules that are still visible in the mex67-5 mutant localize at the nuclear border (Figure 4C). Given that Mex67 has been implicated in the life cycle of diverse RNA classes, we speculate that Mex67 may perform a stabilization function for the poly-A⁺ RNA species as it does for telomerase RNA.

Since old TLC1 molecules underwent at least one round of nucleo-cytoplasmic shuttling, we reasoned that during this shuttling, telomerase RNA must have acquired a property that renders it insensitive to the loss of Mex67. Hence, we asked which telomerase subunits could confer TLC1 stability after export to the cytoplasm. Using a modified inducible TLC1 tagging system in which the new TLC1 RNA carries a tlc1-Sm2T mutation, we demonstrate that TLC1 RNA deficient in Sm₇ binding accumulates in the cytoplasm (Figure 5; Figure 5—figure supplement 1). Given the nuclear instability of the wild type RNA in the absence of Mex67 (see above), we conclude that the Sm₇ complex associates with the TLC1 RNA in the cytoplasm and that this association in turn stabilizes the RNA. Recently a cytoplasmic association with the Sm₇ complex has also been suggested for snRNAs (Becker et al., 2019). Similarly, nuclear export and cytoplasmic Sm₇ binding is also a characteristic of mammalian snRNAs (Fischer et al., 2011).

The data reported here challenge the current view of telomerase biogenesis by placing the Sm₇ binding step after export of telomerase RNA to the cytoplasm (Figure 6). The new model incorporates the idea that in the nucleus, newly synthesized poly-A^- telomerase RNA (Noël et al., 2012) first requires stabilization by Mex67, which may also usher TLC1 to nuclear pores. Whether the Mex67 association occurs co-transcriptionally or in coordination with the 3’-end formation or even after transcript release remains to be determined. Failure to associate with Mex67 results in immediate degradation of new TLC1 transcripts via the nuclear exosome. Next, the RNA is transported to the cytoplasm which absolutely requires Xpo1. This karyopherin does not bind RNA directly and we still do not know what the TLC1 adaptor for Xpo1 is. In this regard, our data are consistent with the possibility that Mex67 itself plays this role in addition to its stabilizing function in the nucleus (Figure 6). If Mex67 indeed acts as an adaptor between TLC1 RNA and Xpo1 exportin, it would likely translocate through the nuclear membrane with the Xpo1-TLC1 complex. Once in the cytoplasm, the TLC1 RNA must rapidly associate with the Sm₇ complex, given that Mex67 will likely disembark from the RNA at the nuclear pore exit and get recycled back into the nucleus (Stewart, 2007). At this stage then, the Sm₇ complex is required for TLC1 RNA stabilization, and in turn, TLC1 stability allows for other telomerase subunits to associate with the maturing RNP. If this step is compromised, TLC1 molecules undergo degradation in the cytoplasm. Whether the cytoplasmic exosome, the Xrn1 nuclease or other machineries perform this function remains to be determined. Eventually, the telomerase RNP is re-imported to the nucleus primarily via Kap122, but alternative and less efficient import pathways do exist (Figure 3C). Finally, our model suggests that the final 5’-TMG capping and 3’-end processing of telomerase RNA occurs in the nucleus after its re-import and is dependent on the presence of the Sm₇-complex, possibly analogous to snRNA maturation (Becker et al., 2019). This final step may thus occur on a relatively mature telomerase RNP, which is entirely feasible given a predicted close proximity of the 5’- and 3’-ends of telomerase RNA (Cech, 2004; Dandjinou et al., 2004). It is also consistent with the fact that active telomerase RNPs only contain 5’-TMG capped telomerase RNAs (Bosoy et al., 2003). Maturation of telomerase RNA may also serve as a final stabilization step for TLC1 RNA and render it insensitive to loss of Mex67, as observed above (Figure 4C).

Our study thus elucidates many of the early events of telomerase RNA biogenesis. However, later stages of TLC1 life cycle remain enigmatic, e.g., does telomerase RNA continue to cycle between the nucleus and the cytoplasm or whether these and final shuttling rounds rely on the same machinery as the initial export and re-import. Although our data show that Mex67 is dispensable for TLC1 stability after the first round of shuttling (Figure 4), it might still act as a TLC1-Xpo1 adaptor for the subsequent rounds of export. Alternatively, TLC1 might use a different adaptor for later shuttling steps or even rely on a different karyopherin (such as Los1 or Msn5).

As in yeast, the generation and stabilization of human telomerase RNA relies on the association with several proteins, including dyskerin, NHP2 and NOP10 as well as factors involved in the non-coding RNA maturation pathway such as PARN (Hoareau-Aveilla et al., 2006; Fu and Collins, 2007; Walne et al., 2007; Moon et al., 2015; Nguyen et al., 2015). Mutations in PARN, dyskerin or NOP10 cause severe genetic disorders, emphasizing the functional significance of these telomerase subunits and RNA maturation mechanisms (Vulliamy et al., 2006; Walne et al., 2007). Furthermore, the loss of telomerase RNA due to PARN mutations is amenable to reversion by small molecules that thus may alleviate the severe associated disease (Brenner and Nandakumar, 2020). However, certain details of human telomerase biogenesis remain enigmatic. For example, how and when do dyskerin and NOP10 associate with telomerase RNA and is association with these subunits needed for RNA processing? Inducible RNA tagging tools as described in this study should greatly aid in teasing out these maturation steps and may provide missing links in human telomerase biogenesis.

Source data files have been uploaded. Strains and materials generated for this study will be freely available on request.

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Author details

1. Yulia Vasianovich

   Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada

   Present address

   Department of Medicine, Faculty of Medicine and Health Sciences, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montreal, Canada

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1643-794X

2. Emmanuel Bajon

   Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada

   Present address

   Department of Biochemistry, Faculty of Medicine, Université de Montréal, Montreal, Canada

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1588-2953

3. Raymund J Wellinger

   Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   Raymund.Wellinger@Usherbrooke.ca

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-6670-2759

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