RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions.

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.

Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields.

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr-Purcell-Meiboom-Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.

m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB.

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.

NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing.

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

Direct evidence for (G)O6···H2-N4(C)+ hydrogen bonding in transient G(syn)-C+ and G(syn)-m5C+ Hoogsteen base pairs in duplex DNA from cytosine amino nitrogen off-resonance R1ρ relaxation dispersion measurements.

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ∼2 Što allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1ρ relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1ρ relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.

Structural and Functional Characterization of Sulfonium Carbon-Oxygen Hydrogen Bonding in the Deoxyamino Sugar Methyltransferase TylM1.

The N-methyltransferase TylM1 from Streptomyces fradiae catalyzes the final step in the biosynthesis of the deoxyamino sugar mycaminose, a substituent of the antibiotic tylosin. The high-resolution crystal structure of TylM1 bound to the methyl donor S-adenosylmethionine (AdoMet) illustrates a network of carbon-oxygen (CH···O) hydrogen bonds between the substrate's sulfonium cation and residues within the active site. These interactions include hydrogen bonds between the methyl and methylene groups of the AdoMet sulfonium cation and the hydroxyl groups of Tyr14 and Ser120 in the enzyme. To examine the functions of these interactions, we generated Tyr14 to phenylalanine (Y14F) and Ser120 to alanine (S120A) mutations to selectively ablate the CH···O hydrogen bonding to AdoMet. The TylM1 S120A mutant exhibited a modest decrease in its catalytic efficiency relative to that of the wild type (WT) enzyme, whereas the Y14F mutation resulted in an approximately 30-fold decrease in catalytic efficiency. In contrast, site-specific substitution of Tyr14 by the noncanonical amino acid p-aminophenylalanine partially restored activity comparable to that of the WT enzyme. Correlatively, quantum mechanical calculations of the activation barrier energies of WT TylM1 and the Tyr14 mutants suggest that substitutions that abrogate hydrogen bonding with the AdoMet methyl group impair methyl transfer. Together, these results offer insights into roles of CH···O hydrogen bonding in modulating the catalytic efficiency of TylM1.

A potentially abundant junctional RNA motif stabilized by m6A and Mg2.

N6-Methyladenosine (m6A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. In addition to recruiting proteins, m6A can modulate RNA function by destabilizing base pairing. Here, we show that when neighbored by a 5' bulge, m6A stabilizes m6A-U base pairs, and global RNA structure by ~1 kcal mol-1. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3' neighbor through a conformation that is stabilized by Mg2+. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m6A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m6A antibodies, both readily recognize m6A in this newly identified motif. The results uncover potentially abundant and functional m6A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data.

Pseudouridine and N6-methyladenosine modifications weaken PUF protein/RNA interactions.

RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications-pseudouridine (Ψ) and N6-methyladenosine (m6A)-affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure. Here, we test the hypothesis that RNA modifications directly affect interactions between RNA-binding proteins and target RNA. We show that Ψ and m6A weaken the binding of the human single-stranded RNA binding protein Pumilio 2 (hPUM2) to its consensus motif, with individual modifications having effects up to approximately threefold and multiple modifications giving larger effects. While there are likely to be some cases where RNA modifications essentially fully ablate protein binding, here we see modest responses that may be more common. Such modest effects could nevertheless profoundly alter the complex landscape of RNA:protein interactions, and the quantitative rather than qualitative nature of these effects underscores the need for quantitative, systems-level accounting of RNA:protein interactions to understand post-transcriptional regulation.

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs.

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

Three RNA Microenvironments Detected in Fluxional Gene Delivery Polyplex Nanoassemblies.

Prototropic and solvatochromatic properties of fluorescein (FL) were employed to detect the presence of microenvironments in polyplexes consisting of polycationic polymer (POCP) and a fluorescein-conjugated RNA, the HIV-1 transactivation response element (TAR-FL). Results reveal new aspects of polyplex structure with respect to polyplex-bound RNA existing in the following local microenvironments: (a) RNA associated with the polyplex that experiences local pH changes in a manner dependent on POCP nitrogen to RNA phosphate ratio (N:P), (b) RNA experiencing relatively acidic local pH environment that remains constant in polyplexes formed after a charge-neutral ratio, and (c) RNA packed close enough to mediate fluorophore/fluorophore quenching. The magnitude of these changes observed as a function of POCP to nucleic acid N:P ratio is polymer dependent. Assessment of the different microenvironments can help elucidate the functional hierarchy of polyplex-bound oligonucleotides and additionally characterize POCPs based on the resulting local pH and solvent properties upon polyplex formation.

Rapid Exchange Between Free and Bound States in RNA-Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release.

A combination of solution NMR, dynamic light scattering (DLS), and fluorescence quenching assays were employed to obtain insights into the dynamics and structural features of a polyplex system consisting of HIV-1 transactivation response element (TAR) and PEGylated generation 5 poly(amidoamine) dendrimer (G5-PEG). NMR chemical shift mapping and (13)C spin relaxation based dynamics measurements depict the polyplex system as a highly dynamic assembly where the RNA, with its local structure and dynamics preserved, rapidly exchanges (

Manipulating unconventional CH-based hydrogen bonding in a methyltransferase via noncanonical amino acid mutagenesis.

Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation's effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

Incorporation of CC steps into Z-DNA: interplay between B-Z junction and Z-DNA helical formation.

The left-handed DNA structure, Z-DNA, is believed to play important roles in gene expression and regulation. Z-DNA forms sequence-specifically with a preference for sequences rich in pyrimidine/purine dinucleotide steps. In vivo, Z-DNA is generated in the presence of negative supercoiling or upon binding proteins that absorb the high energetic cost of the B-to-Z transition, including the creation of distorted junctions between B-DNA and Z-DNA. To date, the sequence preferences for the B-to-Z transition have primarily been studied in the context of sequence repeats lacking B-Z junctions. Here, we develop a method for characterizing sequence-specific preferences for Z-DNA formation and B-Z junction localization within heterogeneous DNA duplexes that is based on combining 2-aminopurine fluorescence measurements with a new quantitative application of circular dichroism spectroscopy for determining the fraction of B- versus Z-DNA. Using this approach, we show that at least three consecutive CC dinucleotide steps, traditionally thought to disfavor Z-DNA, can be incorporated within heterogeneous Z-DNA containing B-Z junctions upon binding to the Zα domain of the RNA adenosine deaminase protein. Our results indicate that the incorporation of CC steps into Z-DNA is driven by favorable sequence-specific Z-Z and B-Z stacking interactions as well as by sequence-specific energetics that localize the distorted B-Z junction at flexible sites. Together, our results expose higher-order complexities in the Z-DNA code within heterogeneous sequences and suggest that Z-DNA can in principle propagate into a wider range of genomic sequence elements than previously thought.

Intrinsic dynamics of DNA-polymer complexes: a mechanism for DNA release.

The transfer of genetic material into cells using nonviral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, line width, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR time scale (

Direct evidence for methyl group coordination by carbon-oxygen hydrogen bonds in the lysine methyltransferase SET7/9.

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and non-histone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH · · · O) hydrogen bonds have been proposed to coordinate the methyl groups of AdoMet and methyllysine within the SET domain active site. However, the presence of these hydrogen bonds has only been inferred due to the uncertainty of hydrogen atom positions in x-ray crystal structures. To experimentally resolve the positions of the methyl hydrogen atoms, we used NMR (1)H chemical shift coupled with quantum mechanics calculations to examine the interactions of the AdoMet methyl group in the active site of the human KMT SET7/9. Our results indicated that at least two of the three hydrogens in the AdoMet methyl group engage in CH · · · O hydrogen bonding. These findings represent direct, quantitative evidence of CH · · · O hydrogen bond formation in the SET domain active site and suggest a role for these interactions in catalysis. Furthermore, thermodynamic analysis of AdoMet binding indicated that these interactions are important for cofactor binding across SET domain enzymes.

Using fluorine nuclear magnetic resonance to probe the interaction of membrane-active peptides with the lipid bilayer.

A variety of biologically active peptides exert their function through direct interactions with the lipid membrane of the cell. These surface interactions are generally transient and highly dynamic, making them hard to study. Here we have examined the feasibility of using solution phase (19)F nuclear magnetic resonance (NMR) to study peptide-membrane interactions. Using the antimicrobial peptide MSI-78 as a model system, we demonstrate that peptide binding to either small unilamellar vesicles (SUVs) or bicelles can readily be detected by simple one-dimensional (19)F NMR experiments with peptides labeled with l-4,4,4-trifluoroethylglycine. The (19)F chemical shift associated with the peptide-membrane complex is sensitive both to the position of the trifluoromethyl reporter group (whether in the hydrophobic face or positively charged face of the amphipathic peptide) and to the curvature of the lipid bilayer (whether the peptide is bound to SUVs or bicelles). (19)F spin echo experiments using the Carr-Purcell-Meiboom-Gill pulse sequence were used to measure the transverse relaxation (T(2)) of the nucleus and thereby examine the local mobility of the MSI-78 analogues bound to bicelles. The fluorine probe positioned in the hydrophobic face of the peptide relaxes at a rate that correlates with the tumbling of the bicelle, suggesting that it is relatively immobile, whereas the probe at the positively charged face relaxes more slowly, indicating this position is much more dynamic. These results are in accord with structural models of MSI-78 bound to lipids and point to the feasibility of using fluorine-labeled peptides to monitor peptide-membrane interactions in living cells.

Modulating protein structure with fluorous amino acids: increased stability and native-like structure conferred on a 4-helix bundle protein by hexafluoroleucine.

There has recently been much interest in exploiting the unusual properties associated with fluorocarbons to modulate the physicochemical properties of proteins. Here we present a detailed investigation into the effect on structure and stability of systematically repacking the hydrophobic core of a model protein with the extensively fluorinated (fluorous) amino acid l-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). The starting point was a 27-residue peptide, alpha(4)-H, that adopts an antiparallel 4-alpha-helix bundle structure, and in which the hydrophobic core comprises six layers of leucine residues introduced at the "a" and "d" positions of the canonical heptad repeat. A series of peptides were synthesized in which the central two (alpha(4)-F(2))(,) four (alpha(4)-F(4)), or all six layers (alpha(4)-F(6)) of the core were substituted hFLeu. The free energy of unfolding increases by 0.3 (kcal/mol)/hFLeu on repacking the central two layers and by an additional 0.12 (kcal/mol)/hFLeu on repacking additional layers, so that alpha(4)-F(6) is approximately 25% more stable than the nonfluorinated protein alpha(4)-H. One-dimensional proton, two-dimensional (1)H-(15)N HSQC, and (19)F NMR spectroscopies were used to examine the effect of fluorination on the conformational dynamics of the peptide. Unexpectedly, increasing the degree of fluorination also appears to result in peptides that possess a more structured backbone and less fluid hydrophobic core. The latter only occurs in alpha(4)-F(4) and alpha(4)-F(6), suggesting that crowding of the hFLeu residues may restrict the amplitude and/or time scales for rotation of the side chains.

Insight into the CSA tensors of nucleobase carbons in RNA polynucleotides from solution measurements of residual CSA: towards new long-range orientational constraints.

Using residual chemical shift anisotropies (RCSAs) measured in a weakly aligned stem-loop RNA, we examined the carbon chemical shift anisotropy (CSA) tensors of nucleobase adenine C2, pyrimidine C5 and C6, and purine C8. The differences between the measured RCSAs and the values back-calculated using three nucleobase carbon CSA sets [D. Stueber, D.M. Grant, 13C and 15N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551; D. Sitkoff, D.A. Case, Theories of chemical shift anisotropies in proteins and nucleic acids, Prog. NMR Spectrosc. 32 (1998) 165-190; R. Fiala, J. Czernek, V. Sklenar, Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids, J. Biomol. NMR 16 (2000) 291-302] reported previously for mononucleotides (1.4 Hz) is significantly smaller than the predicted RCSA range (-10-10 Hz) but remains larger than the RCSA measurement uncertainty (0.8 Hz). Fitting of the traceless principal CSA values to the measured RCSAs using a grid search procedure yields a cytosine C5 CSA magnitude (CSAa=(3/2.(delta11(2)+delta22(2)+delta33(2)))1/2=173+/-21 ppm), which is significantly higher than the reported mononucleotide values (131-138 ppm) and a guanine C8 CSAa (148+/-13 ppm) that is in very good agreement with the mononucleotide value reported by solid-state NMR [134 ppm, D. Stueber, D.M. Grant, 13C and (15)N chemical shift tensors in adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine, J. Am. Chem. Soc. 124 (2002) 10539-10551]. Owing to a unique sensitivity to directions normal to the base plane, the RCSAs can be translated into useful long-range orientational constraints for RNA structure determination even after allowing for substantial uncertainty in the nucleobase carbon CSA tensors.