RNA–protein binding interface in the telomerase ribonucleoprotein

Abstract

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR-TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA-protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain.

Telomerase and retrotransposons: Reverse transcriptases that shaped genomes

“…Additionally, on the basis of the structure of the Tribolium castaneum TERT, she proposed two plausible views of the relative disposition of the core domain and TERT. A parallel advance on the STE is reported by Julian Chen and colleagues in an accompanying article (23). Using a unique photoaffinity cross-linking approach and mass spectrometry, they identified three nucleotideamino acid contacts between a vertebrate STE (known as CR4/5) and the corresponding TERT, thus bringing into sharper focus the architecture of the telomerase RNP.…”

Structure and function of echinoderm telomerase RNA

“…Within the vertebrate CR4/5 domain, the P6 stem-loop contains a conserved single-nucleotide uridine bulge (Chen et al 2000;Xie et al 2008). The deletion of this uridine bulge drastically reduces binding of the medaka fish CR4/5 domain to its TERT protein (Bley et al 2011). To investigate whether either single-nucleotide bulge in purple sea urchin eCR4/5 has a similar vital function for RNA-protein interaction, we generated single-residue deletion mutants of P4.2-Δ2-mini: ΔC345 and ΔU392 (Fig.…”

Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach

“…Functioning as a ribonucleoprotein (RNP) enzyme, telomerase consists of two essential core components: the catalytic telomerase reverse transcriptase (TERT) protein and telomerase RNA (TR) that provides the template for DNA synthesis (Blackburn and Collins 2011). TERT is highly conserved, with the central domain constituting the active site and an N-terminal domain that binds specifically to the TR (Bley et al 2011;Mason et al 2011). In contrast, TR is extremely divergent in size, sequence, and structure (Podlevsky et al 2008;Podlevsky and Chen 2012).…”