New perspectives on telomerase RNA structure and function

Abstract

Telomerase is an ancient ribonucleoprotein (RNP) that protects the ends of linear chromosomes from the loss of critical coding sequences through repetitive addition of short DNA sequences. These repeats comprise the telomere, which together with many accessory proteins, protect chromosomal ends from degradation and unwanted DNA repair. Telomerase is a unique reverse transcriptase (RT) that carries its own RNA to use as a template for repeat addition. Over decades of research, it has become clear that there are many diverse, crucial functions played by telomerase RNA beyond simply acting as a template. In this review, we highlight recent findings in three model systems: ciliates, yeast and vertebrates, that have shifted the way the field views the structural and mechanistic role(s) of RNA within the functional telomerase RNP complex. Viewed in this light, we hope to demonstrate that while telomerase RNA is just one example of the myriad functional RNA in the cell, insights into its structure and mechanism have wide-ranging impacts. WIREs RNA 2018, 9:e1456. doi: 10.1002/wrna.1456 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Figure 1. Telomerase Subunits and Catalytic Mechanism

(A) Conserved structural elements of telomerase RNA (TR) secondary structural models from Ciliates, Vertebrates, and Yeasts are shown. The template boundary element (TBE) (red), template (yellow), RNA pseudoknot (PK) fold (blue), and stem terminus element (green) are indicated for each organism. A dashed line indicates regions that are hyper-variable between species within each category. (B) Domain organization of telomerase reverse transcriptase (TERT) catalytic protein subunit from Ciliates, Yeasts, and Vertebrates. The conserved essential N-terminal domain (TEN), RNA binding domain (RBD), reverse transcriptase (RT) domain, and C-terminal element CTE) are all indicated. In addition, specific sequence motifs that are evolutionarily conserved and have been shown to be important for telomerase function are indicated. (C) Cartoon schematic of the Tetrahymena telomerase catalytic cycle. The 3’ end of a single-stranded DNA substrate (green) binds in the active site of the telomerase complex and aligns with the RNA template through Watson-Crick base pairing. This provides a short RNA/DNA hybrid that serves as the substrate for the catalytic RT domain within TERT to extend the telomere DNA using the integral telomerase RNA template. Upon reaching the template boundary, the newly formed RNA/DNA hybrid must dissociate and realign with the downstream region of the telomerase RNA template to support telomere DNA repeat addition processivity (RAP). For clarity, color-coding throughout the review article is consistent with the color scheme established in this figure.

Figure 2. RNA structure and function in ciliate telomerase

(A) High-resolution structure of the Tetrahymena p65 protein xRRM protein domain bound to stem-loop IV of telomerase RNA. Figure adapted from (36) PDB 4ERD. (B) High-resolution structure of the Tetrahymena TERT-RBD domain bound to the base of stem-loop II, comprising the template boundary definition complex. Figure adapted from (37) PDB 5C9H. (C) High-resolution structure of the Tetrahymena RNA pseudoknot domain. Figure adapted from (38) PDB 5KMZ. (D) Schematic model of Tetrahymena telomerase RNA organization based upon the medium-resolution structure of the complete holoenzyme solved by cryo-electron microscopy. Figure adapted from (39). (E) Cartoon model for reorganization of Tetrahymena RNA pseudoknot fold upon binding and assembly with TERT protein subunit. (F) The RNA accordion model for RNA structural rearrangements during telomerase catalysis. During telomere DNA repeat synthesis the RNA regions flanking each side of the template undergo compaction and expansion to facilitate movement of the template through the RT active site.

Figure 3. RNA structure and function in yeast telomerase

(A) Schematic model of the core domain of yeast telomerase RNA, highlighting the area of required connectivity (ARC) outlined in dashed black box. Disruption of the RNA backbone in this region of the telomerase RNA disrupts catalytic activity. (B) High-resolution structure of the RNA pseudoknot fold from the budding yeast Kluyveromyces lactis. Figure is adapted from (56) PDB 2M8K. (C) Model for partial splicing during maturation of fission yeast telomerase RNA. After recognition by the yeast spliceosome, the first step of splicing produces a functional telomerase RNA with a mature 3’ end. The second step of splicing is highly inefficient and usually abortive for this transcript, but when it does proceed, the splicing product is an inactive telomerase RNA that is quickly degraded. (D) Model for active site ‘stuttering’ during yeast telomere DNA repeat synthesis. Many species of yeast possess irregular telomere repeat sequences. In the ‘Stuttering Model’, efficient template boundary definition is reinforced by the distal stem terminus element (STE), preventing run on reverse transcription beyond the template. After realignment of the 3’ end of the DNA substrate, the RT active site can incorporate extra dGTP nucleotides, resulting in telomere repeats of varying length and sequence.

Figure 4. RNA structure and function in vertebrate telomerase

(A) High-resolution structure of a minimal human telomerase RNA pseudoknot was the first to reveal the conserved base triples that stabilize the RNA fold. Figure based on (64) PDB 2K95. (B) Model for the human telomerase RNA core domain, which include the template and RNA pseudoknot fold. The model predicts a triangular organization of the RNA which is compatible with available structural data for the TERT protein subunit. Figure adapted from (66). (C) Structural analysis of the conserved region 4/5 (CR4/5) from the Medaka fish. This RNA fragment contains the essential STE (P6.1 in vertebrates) required for telomerase catalysis. High-resolution structures of this RNA domain bound to the TERT-RBD or in the absence of protein demonstrate the large scale structural reorganization of the three-way junction upon RNP assembly. Figures based upon (71, 74) PDB 4O26 and PDB 2MHI. (D) Model for the self-regulating RNA template in human telomerase. Biochemical mutagenesis analysis demonstrated the presence of a pause signal in the nascent RNA/DNA hybrid. This A-T base pair induces a kinetic pause that serves to reinforce template boundary definition and promotes translocation of the DNA product. (E) RNA pseudoknot tracking model for human telomerase catalysis. Biophysical and computational modeling studies, using a combination of single molecule FRET measurements paired with ROSETTA based molecular modeling, revealed the human RNA pseudoknot fold exhibits a large-scale conformational rearrangement during telomerase catalysis. The proximity of the RNA pseudoknot to the TERT CTE domain, which represents the polymerase ‘thumb’ domain, suggests movement of the pseudoknot may serve to stabilize a conformation of the RT active site required for repeat addition processivity.