Structural study of elements of <i>Tetrahymena</i> telomerase RNA stem–loop IV domain important for function

Richards, R. J.; Wu, Huixian; Trantı́rek, Lukáš; O’Connor, Catherine; Collins, Kathleen; Feigon, Juli

doi:10.1261/rna.112306

Cited by 40 publications

(49 citation statements)

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“…1A) also reduced NAP, while substitution of other loop positions reduced repeat addition processivity (RAP) or activity overall (Sperger and Cech 2001;Cunningham and Collins 2005;O'Connor et al 2005). NMR structures of stem-loop IV revealed that loop nucleotide U138 forms a noncanonical base pair with C132, which organizes stacking of all other bases in the loop except the flipped-out and relatively unconstrained U137 (Chen et al 2006;Richards et al 2006). This structure suggests a rationale for the functional significance of U137 and for the importance of the U138 in ejecting U137 from the constraints of base stacking.…”

Section: Resultsmentioning

confidence: 99%

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability

Robart

O’Connor²,

Collins³

2010

RNA

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Telomerase adds simple-sequence repeats to chromosome 39 ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

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Section: Resultsmentioning

confidence: 99%

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability

Robart

O’Connor²,

Collins³

2010

RNA

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“…Here, the derived motional amplitudes will underestimate the real motional amplitudes 24 . Simulations (data not shown) as well as experimental results show that differences on the order of three base pairs can be sufficient to take an RNA system outside the extreme coupling limit and into the intermediate regime 21,22,24,38,54,55 . Extensive elongation of helices using isotopic labeling strategies that render elongation residues invisible can also be used to bring the RNA system closer to within the decoupling limit 56 .…”

Section: Experimental Designmentioning

confidence: 98%

“…The protocol in its current form yields 4 n−1 degenerate solutions for orienting n helices and this degeneracy has to be lifted with the use of other experimental or geometrical restraints. Note that many variants of this protocol, some of which allow simultaneous local structure determination of molecular fragments, have also been described [34][35][36][37][38] .…”

Section: Introductionmentioning

confidence: 99%

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

et al. 2007

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We present a protocol for determining the relative orientation and dynamics of A-form helices in 13 C/ 15 N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

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“…10,11,15,16 Some mutations in TER and TERT that abrogate or decrease activity in telomerase assembled in vitro from TERT and TER are rescued when p65 is present, which further underscores the essential role of p65 in the biogenesis and assembly of the telomerase holoenzyme. 15,17 The interaction of p65 with TER is discussed next in detail.…”

Section: Introductionmentioning

confidence: 99%

“…26 (C) Model of teR SL4 bound to p65 xRRM2. the model is based on p65 xRRM2 bound to teR S4 (PDB iD 4eRD), 13 the loop 4 is taken from the solution structure (PDB iD 2M21) 17 and placed on the distal stem 4.…”

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confidence: 99%

xRRM

Singh¹,

Choi

Feigon³

2013

RNA Biology

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Genuine La and La-related proteins group 7 (LARP7) bind to the non-coding RNAs transcribed by RNA polymerase III (RNAPIII), which end in UUU-3'OH. The La motif and RRM1 of these proteins (the La module) cooperate to bind the UUU-3'OH, protecting the RNA from degradation, while other domains may be important for RNA folding or other functions. Among the RNAPIII transcripts is ciliate telomerase RNA (TER). p65, a member of the LARP7 family, is an integral Tetrahymena thermophila telomerase holoenzyme protein required for TER biogenesis and telomerase RNP assembly. p65, together with TER and telomerase reverse transcriptase (TERT), form the Tetrahymena telomerase RNP catalytic core. p65 has an N-terminal domain followed by a La module and a C-terminal domain, which binds to the TER stem 4. We recently showed that the p65 C-terminal domain harbors a cryptic, atypical RRM, which uses a unique mode of single-and doublestrand RNA binding and is required for telomerase RNP catalytic core assembly. This domain, which we named xRRM, appears to be present in and unique to genuine La and LARP7 proteins. Here we review the structure of the xRRM, discuss how this domain could recognize diverse substrates of La and LARP7 proteins and discuss the functional implications of the xRRM as an RNP chaperone.

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Structural study of elements of Tetrahymena telomerase RNA stem–loop IV domain important for function

Cited by 40 publications

References 50 publications

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

xRRM

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