RNA-protein binding interface in the telomerase ribonucleoprotein

Christopher J. Bley, Xiaodong Qi, Dustin P. Rand, Chad Borges, Randall W. Nelson, Julian Chen

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)20333-20338
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number51
DOIs
StatePublished - Dec 20 2011

Fingerprint

Ribonucleoproteins
RNA-Binding Proteins
Telomerase
RNA
Oryzias
Tetrahymena
Proteins
Structural Models
RNA-Directed DNA Polymerase
Vertebrates
Mass Spectrometry
Carrier Proteins
Hydrolysis
Amino Acids

ASJC Scopus subject areas

  • General

Cite this

RNA-protein binding interface in the telomerase ribonucleoprotein. / Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad; Nelson, Randall W.; Chen, Julian.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, No. 51, 20.12.2011, p. 20333-20338.

Research output: Contribution to journalArticle

Bley, Christopher J. ; Qi, Xiaodong ; Rand, Dustin P. ; Borges, Chad ; Nelson, Randall W. ; Chen, Julian. / RNA-protein binding interface in the telomerase ribonucleoprotein. In: Proceedings of the National Academy of Sciences of the United States of America. 2011 ; Vol. 108, No. 51. pp. 20333-20338.
@article{52929ef9714d489f9931a8ab2f2cb9b8,
title = "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.",
author = "Bley, {Christopher J.} and Xiaodong Qi and Rand, {Dustin P.} and Chad Borges and Nelson, {Randall W.} and Julian Chen",
year = "2011",
month = "12",
day = "20",
doi = "10.1073/pnas.1100270108",
language = "English (US)",
volume = "108",
pages = "20333--20338",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "51",

}

TY - JOUR

T1 - RNA-protein binding interface in the telomerase ribonucleoprotein

AU - Bley, Christopher J.

AU - Qi, Xiaodong

AU - Rand, Dustin P.

AU - Borges, Chad

AU - Nelson, Randall W.

AU - Chen, Julian

PY - 2011/12/20

Y1 - 2011/12/20

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84862908568&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862908568&partnerID=8YFLogxK

U2 - 10.1073/pnas.1100270108

DO - 10.1073/pnas.1100270108

M3 - Article

VL - 108

SP - 20333

EP - 20338

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 51

ER -