Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Nobles, Kelly N.; Yarian, Connie S.; Liu, Guihua; Guenther, Richard; Agris, Paul F.

doi:10.1093/nar/gkf595

Cited by 69 publications

(66 citation statements)

References 59 publications

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“…In this way, Ψ's can stabilize tRNA folding and increase the fidelity of codon-anticodon interactions, stabilize certain stem-loop conformations of snRNAs, and enable ribosome assembly (Yarian et al 1999;Greenbaum 2001, 2002b;Nobles et al 2002;Cabello-Villegas and Nikonowicz 2005;Denmon et al 2011;Jack et al 2011;Penzo et al 2015). The majority of indications that pseudouridylation plays an important biological role stems indirectly from the observation that Ψ's are modulated in response to different cellular conditions.…”

Section: Introductionmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

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Section: Introductionmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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“…Second, tRNA has a well-folded native tertiary structure that is relevant to many functional RNAs in eukaryotic and prokaryotic cells. Third, the T7 transcript of tRNA has similar properties as the naturally occurring modified RNA (Sampson and Uhlenbeck 1988;Nobles et al 2002;; Whitman 2011) but is easily mutated to provide weakened tertiary structure so that effects of molecular crowders and cosolutes on secondary and tertiary structure individually can be assessed (Fig. 1B).…”

Section: Introductionmentioning

confidence: 99%

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Strulson

Boyer

Whitman

et al. 2014

RNA

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Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg 2+ ion concentrations are low, K + concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg 2+ (0.5-2 mM) and K + (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperaturedependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

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“…Yet, at the atomic level little is known concerning the effects of the many modifications on hydrogen bonding and basepairing. Many of the more than 100 modified nucleotide chemistries of RNA are complex, involved in hydrogen bonding, and thus important to RNA folding, structure, dynamics, and function (Agris 1996;Nobles et al 2002;Gustilo et al 2008). Thus, a detailed understanding of the geometries and energies of hydrogen bonding by modified nucleotides is applicable to the analysis of native RNAs and to the future design of novel RNAs with new functions.…”

Section: Introductionmentioning

confidence: 99%

Free energy calculation of modified base-pair formation in explicit solvent: A predictive model

Vendeix

Munoz²,

Agris³

2009

RNA

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The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between different RNAs, especially in formation of mismatch base pairs. Thus, an understanding of the geometry and strength of the base-pairing of modified ribonucleoside 59-monophosphates, previously not defined, is applicable to investigations of RNA structure and function and of the design of novel RNAs. The geometry and free energies of base-pairings were calculated in aqueous solution under neutral conditions with AMBER force fields and molecular dynamics simulations (MDSs). For example, unmodified uridines were observed to bind to uridine and cytidine with significant stability, but the ribose C19-C19 distances were far short (;8.9 Å ) of distances observed for canonical A-form RNA helices. In contrast, 5-oxyacetic acid uridine, known to bind adenosine, wobble to guanosine, and form mismatch base pairs with uridine and cytidine, bound adenosine and guanosine with geometries and energies comparable to an unmodified uridine. However, the 5-oxyacetic acid uridine base paired to uridine and cytidine with a C19-C19 distance comparable to that of an A-form helix, ;11 Å , when a H 2 O molecule migrated between and stably hydrogen bonded to both bases. Even in formation of canonical base pairs, intermediate structures with a second energy minimum consisted of transient H 2 O molecules forming hydrogen bonded bridges between the two bases. Thus, MDS is predictive of the effects of modifications, H 2 O molecule intervention in the formation of base-pair geometry, and energies that are important for native RNA structure and function.

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Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Cited by 69 publications

References 59 publications

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Free energy calculation of modified base-pair formation in explicit solvent: A predictive model

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