RNA Binding Domain of Telomerase Reverse Transcriptase

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes. The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA. Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telomerase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity. In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT. Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs. The minimalTetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species. With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well. Finally, we defined theTetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide region 5′ of the template is critical for TERT binding and that the 5′ end of telomerase RNA is sufficient for TERT binding. Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template.

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The PUMILIO−RNA Interaction: A Single RNA-Binding Domain Monomer Recognizes a Bipartite Target Sequence†

Biochemistry ◽

10.1021/bi982264s ◽

1999 ◽

Vol 38 (2) ◽

pp. 596-604 ◽

Cited By ~ 51

Author(s):

Phillip D. Zamore ◽

David P. Bartel ◽

Ruth Lehmann ◽

James R. Williamson

Keyword(s):

Rna Binding ◽

Binding Domain ◽

Target Sequence ◽

Rna Binding Domain ◽

Rna Interaction

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LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain

Nucleic Acids Research ◽

10.1093/nar/gkaa652 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9262-9272 ◽

Cited By ~ 1

Author(s):

Deqiang Ding ◽

Chao Wei ◽

Kunzhe Dong ◽

Jiali Liu ◽

Alexander Stanton ◽

...

Keyword(s):

Rna Binding ◽

Specific Interaction ◽

Binding Activity ◽

Binding Domain ◽

Binding Property ◽

Domain Family ◽

Rna Sequences ◽

G Quadruplex ◽

Rna Binding Domain ◽

Rna Interaction

Abstract LOTUS domains are helix-turn-helix protein folds identified in essential germline proteins and are conserved in prokaryotes and eukaryotes. Despite originally predicted as an RNA binding domain, its molecular binding activity towards RNA and protein is controversial. In particular, the most conserved binding property for the LOTUS domain family remains unknown. Here, we uncovered an unexpected specific interaction of LOTUS domains with G-rich RNA sequences. Intriguingly, LOTUS domains exhibit high affinity to RNA G-quadruplex tertiary structures implicated in diverse cellular processes including piRNA biogenesis. This novel LOTUS domain-RNA interaction is conserved in bacteria, plants and animals, comprising the most ancient binding feature of the LOTUS domain family. By contrast, LOTUS domains do not preferentially interact with DNA G-quadruplexes. We further show that a subset of LOTUS domains display both RNA and protein binding activities. These findings identify the LOTUS domain as a specialized RNA binding domain across phyla and underscore the molecular mechanism underlying the function of LOTUS domain-containing proteins in RNA metabolism and regulation.

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A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.00239-20 ◽

2020 ◽

Vol 40 (24) ◽

Author(s):

Melissa A. Mefford ◽

Evan P. Hass ◽

David C. Zappulla

Keyword(s):

Reverse Transcriptase ◽

Rna Binding ◽

Telomerase Reverse Transcriptase ◽

Genome Replication ◽

Telomerase Rna ◽

Protein Subunit ◽

Binding Interaction ◽

Catalytic Core

ABSTRACT The telomerase ribonucleoprotein (RNP) counters the chromosome end replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (telomerase reverse transcriptase [TERT]). Although the majority of the 1,157-nucleotide (nt) Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4 bp of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas the ΔCEH and 1- and 2-bp alleles do not support telomerase function. Using the CRISPR/nuclease-deactivated Cas9 (dCas9)-based CARRY (CRISPR-assisted RNA–RNA-binding protein [RBP] yeast) two-hybrid assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nt that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices suggest that this binding interaction with TERT is dictated more by secondary than by primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

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Functional Multimerization of Human Telomerase Requires an RNA Interaction Domain in the N Terminus of the Catalytic Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.22.4.1253-1265.2002 ◽

2002 ◽

Vol 22 (4) ◽

pp. 1253-1265 ◽

Cited By ~ 74

Author(s):

Tara J. Moriarty ◽

Sylvain Huard ◽

Sophie Dupuis ◽

Chantal Autexier

Keyword(s):

Reverse Transcriptase ◽

Rna Binding ◽

Catalytic Subunit ◽

Binding Motif ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Interaction Domain ◽

N Terminus ◽

Human Telomerase ◽

Rna Interaction

ABSTRACT Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

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The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain

Nucleic Acids Research ◽

10.1093/nar/gkaa548 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7981-7990

Author(s):

Laura Carole Keffer-Wilkes ◽

Emily F Soon ◽

Ute Kothe

Keyword(s):

Catalytic Activity ◽

Rna Binding ◽

Binding Domain ◽

Rna Modification ◽

Functional Link ◽

Pseudouridine Synthase ◽

Rna Binding Domain ◽

Mechanistic Basis ◽

Trna Folding

Abstract tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA’s D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA’s tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

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