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Telomerase RNA Biosynthesis and Processing ISSN 0006-2979, Biochemistry (Moscow), 2012, Vol. 77, No. 10, pp. 1120-1128. © Pleiades Publishing, Ltd., 2012. Published in Russian in Biokhimiya, 2012, Vol. 77, No. 10, pp. 1350-1361. REVIEW Telomerase RNA Biosynthesis and Processing E. M. Smekalova1, O. S. Shubernetskaya1, M. I. Zvereva1,2*, E. V. Gromenko1, M. P. Rubtsova1,2, and O. A. Dontsova1,2 1Chemical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia 2Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; E-mail: [email protected] Received April 13, 2012 Abstract—Telomerase synthesizes repetitive G-rich sequences (telomeric repeats) at the ends of eukaryotic chromosomes. This mechanism maintains the integrity of the genome, as telomere shortening leads to degradation and fusion of chromo- somes. The core components of telomerase are the telomerase catalytic subunit and telomerase RNA, which possesses a small template region serving for the synthesis of a telomeric repeat. Mutations in the telomerase RNA are associated with some cases of aplastic anemia and also cause dyskeratosis congenita, myelodysplasia, and pulmonary fibrosis. Telomerase is active in 85% of cancers, and telomerase activation is one of the first steps in cell transformation. The study of telomerase and pathways where this enzyme is involved will help to understand the mechanism of the mentioned diseases and to devel- op new approaches for their treatment. In this review we describe the modern conception of telomerase RNA biosynthesis, processing, and functioning in the three most studied systems – yeast, vertebrates, and ciliates. DOI: 10.1134/S0006297912100045 Key words: telomerase RNA, telomerase, telomeres Telomeres are specialized DNA–protein structures RNA/DNA duplex to allow for synthesis of the next that are localized at the ends of eukaryotic chromosomes; repeat (Fig. 1). Sequential synthesis of the repetitive their main function is to maintain stability of the genome sequences on the basis of one single matrix is not an [1]. Telomeric DNA consists of repetitive sequences inherent property of usual polymerases, and it demands a (telomeric repeats). The classical replication mechanism specific mechanism to release the telomerase RNA cannot provide synthesis of a chromosomal end; that matrix region after each round of replication. The reac- leads to telomere shortening after each round of cell divi- tion starts from 3′-end telomere binding to the region sion, destabilization of the genome, and senescence. To downstream of the matrix, which results in formation of a prevent those processes, cell uses different mechanisms, short hybrid DNA/RNA duplex and then proceeds with the main being telomerase activation. Telomerase synthe- the synthesis of one telomeric repeat. In case of human sizes long sequences that consist of telomeric repeats telomerases, six nucleotides are reverse transcribed into using a small part of telomerase RNA as a matrix for the 5′-GGTTAG-3′ sequence [4]. After reaching the end telomere synthesis [2]. The telomerase catalytic subunit of the matrix region, synthesis stops; newly synthesized (TElomerase Reverse Transcriptase, TERT) and DNA can either translocate to the beginning of the matrix Telomerase RNA (TR) thus constitute a core enzyme that region or dissociate from the enzyme. Translocation is can maintain in vitro telomerase activity in the recon- known to be a complicated multistage process; its mech- struction system, while in vivo there are many more fac- anism is poorly understood even now, after decades of tors needed to provide proper functioning of the enzyme extensive studies of telomerase. Translocation involves [3]. Thus telomerase is an RNA-dependent DNA poly- several stages: unwinding of the DNA/RNA heterodu- merase, which acts as a reverse transcriptase on the basis plex, relocation, and binding of the DNA to the begin- of its own RNA component. ning of the template [5]. The ability to synthesize several The telomerase catalytic cycle consists of two stages: repeats in a row is termed telomerase processivity. Its effi- synthesis of a telomeric repeat on the 3′ terminus of a ciency depends on various factors [6]. telomere, and then release of the matrix from the Telomerase structure and function, TERT in partic- ular, were discussed in detail in a recent review [7]. Here * To whom correspondence should be addressed. we concentrate on the synthesis and maturation of the 1120 TELOMERASE RNA BIOSYNTHESIS AND PROCESSING 1121 Fig. 1. The telomerase reaction cycle. The nucleotide sequence is specified for human telomerase. second component of the telomerase complex – telom- domain (trans-activating domain) and TERT allows erase RNA. Its functions are not restricted to providing reconstruction of the active complex. The other elements the template for telomeric repeat synthesis. Different of TR high order structure are specific for particular parts of the complicated molecule structure shape the classes of organisms. For example, the presence of the telomerase catalytic center together with amino acids of Est1 binding hairpin in yeast TRs [21] or the presence of TERT, participate in the catalytic reaction of the H/ACA scaRNA (small Cajal body RNA) domain in the nucleotide incorporation, mediate efficiency of the 3′ region of vertebrate TRs [22]. translocation process [8], condition the association of the Stable accumulation of most noncoding cellular different telomerase protein factors and complex assem- RNAs implies processing of the initial transcript. This bly [9, 10], and play a key role in transport [11, 12] and can involve RNA splicing, modification of nucleotide regulation of telomerase activity [13]. base and carbohydrate skeleton, and association with Telomerase RNAs of different organisms vary great- proteins, which stabilize the molecule to prevent action of ly in terms of structure, sequence, and length. The length nonspecific nucleases. We will further look closer at the of ciliate TR is 147-205 nt, vertebrate TR ranges from 312 formation of the initial transcript and TR processing for to 559 nt, and yeast ranges from 779 to 2030 nt [14, 15]. the three most studied groups of telomerase RNA – for Comparative and phylogenetic analysis of TRs together yeast, human (which represents vertebrates), and ciliates. with structural studies has been used to predict the 2D structure of TR for ciliates and vertebrates [9, 16]. For yeast telomerase RNA, this was a more difficult task due TRANSCRIPTION AND PROCESSING to the large length and evolutionary flexibility of the mol- ecule. However, it has been shown that a large part of Transcription and processing of yeast telomerase RNA yeast TR is not required for the functioning of the com- (Fig. 2a). Studies of model systems showed that yeast TR plex [17]. Studies have revealed several elements of high is transcribed by RNA-polymerase II, capped by order structure that are conserved between telomerase monomethylguanosine (m7G), and polyadenylated [23]. RNAs of different species. In addition to the template During maturation of the molecule, a polyadenylic tail is region, TRs contain the Template Boundary Element removed and TR is capped by 2,2,7-trimethylguanosine (TBE) situated from the 5′ terminus of the template. It (TMG) [24]. Telomerase RNA of Saccharomyces cerevisiae restricts the reaction of reverse transcription in such a way (TLC1) is stabilized by association with Sm proteins that telomerase would not be able to synthesize sequence from its 3′ end. The function of Sm proteins is to protect beyond the template [18, 19]. Also, one can point out a and stabilize the 3′ end of snRNAs (small nuclear RNAs) large region that includes template, pseudoknot, and a [24]. They function as RNA chaperons and can facilitate hairpin end [20]. In vertebrates this structure is known as modification or degradation of the RNA, participate in the core domain, which, together with the CR4/CR5 intracellular transport, and stabilize the RNA molecule BIOCHEMISTRY (Moscow) Vol. 77 No. 10 2012 1122 SMEKALOVA et al. [25]. The complex functions as a multimer that gathers region of TLC1 contains an AATAA sequence, which cor- from separately associated SmD1-D2, Sm-E-F-G, and responds to one of the two conserved initiation sites used SmB-D3. The primary structure of the vast majority of by polymerase II in S. cerevisiae. TR mRNA holds several TRs includes the Sm protein binding site AU5-6GPu, cis-elements that provide high efficiency of polyadenyla- which is identical to the Sm site of U4 snRNA. Mutations tion. In S. cerevisiae, those elements are A-rich position- in this region of TR result in a cell being unable to process ing element, A–U rich element enhancing the efficiency the 3′ end of the molecule, accumulation of the of the first element, and a polyadenylation site consisting polyadenylated form of TR, and transcript degradation of pyrimidines and adenosines (YA). Mutations in the sug- [24]. However, cells remain viable, but telomeres shorten gested cis-elements did not decrease cell viability, but the in the strains bearing mutations in the Sm site [26]. It was position of polyadenylation was changed and the amount shown that at least 77% of active telomerase interacts with of TR polyadenylated form decreased [23]. Recent data Sm proteins [24]. Co-immunoprecipitation revealed that indicate that the polyadenylation process does not partici- TR is associated with two out of seven Sm proteins – pate in producing the functional TR transcript,
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