In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Platelets subvert T cell immunity against cancer via GARP-TGFß axis.

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor ß (TGFß) and lactate as major platelet-derived soluble factors to obliterate CD4+ and CD8+ T cell functions. Moreover, we found that platelets are the dominant source of functional TGFß systemically as well as in the tumor microenvironment through constitutive expression of the TGFß-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFß per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFß activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFß axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer.

Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype.

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration, and survival in many cell types. Here, the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: noninvasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt, and metM-Wntliver cells, 12, 29, and 25 metabolites, respectively, were significantly different (P < 0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and ß-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration, and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype. IMPLICATIONS: Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189-200. ©2016 AACR.

Structure Calculation and Reconstruction of Discrete-State Dynamics from Residual Dipolar Couplings.

Residual dipolar couplings (RDCs) acquired by nuclear magnetic resonance (NMR) spectroscopy are an indispensable source of information in investigation of molecular structures and dynamics. Here, we present a comprehensive strategy for structure calculation and reconstruction of discrete-state dynamics from RDC data that is based on the singular value decomposition (SVD) method of order tensor estimation. In addition to structure determination, we provide a mechanism of producing an ensemble of conformations for the dynamical regions of a protein from RDC data. The developed methodology has been tested on simulated RDC data with ±1 Hz of error from an 83 residue α protein (PDB ID 1A1Z ) and a 213 residue α/ß protein DGCR8 (PDB ID 2YT4 ). In nearly all instances, our method reproduced the structure of the protein including the conformational ensemble to within less than 2 Å. On the basis of our investigations, arc motions with more than 30° of rotation are identified as internal dynamics and are reconstructed with sufficient accuracy. Furthermore, states with relative occupancies above 20% are consistently recognized and reconstructed successfully. Arc motions with a magnitude of 15° or relative occupancy of less than 10% are consistently unrecognizable as dynamical regions within the context of ±1 Hz of error.

¹9F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR.

Naturally occurring RNA lacks fluorine-19 ((19)F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, (19)F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of (19)F include high NMR sensitivity (83% of (1)H), high natural abundance (100%), and the extreme sensitivity of (19)F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5'-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase.

(1)H, (15)N and (13)C backbone resonance assignments of the N-terminal, tandem KH domains of human hnRNP E1.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) can be divided into subgroups based on their RNA-binding characteristics. One subgroup in mammalian cells are the Poly(C)-binding proteins (PCBPs) comprised of hnRNP K/J and hnRNP E1-4 [the latter also known as PCBP 1-4 or α-complex proteins (α-CP) 1-4]. Each subgroup member has three K homology (KH) nucleic acid-binding domains. Individual KH domains bind short single-stranded (ss), poly-pyrimidine-rich nucleic acid sequences with rather weak affinity. In this study, we report the (1)H, (13)C and (15)N backbone resonance assignments of the first and second KH domains of hnRNP E1, which plays a pivotal role in posttranscriptional and translational regulation of RNA targets. Our NMR assignments lay the foundation for a detailed investigation of the dynamic cooperation of the tandem KH1 and KH2 domains to bind nucleic acids.

Development of inhibitors of heterotrimeric Gαi subunits.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gßγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gαi-GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

Multivalent Amino Sugars to Recognize Different TAR RNA Conformations.

Neomycin dimers synthesized using "click chemistry" with varying functionality and length in the linker region have been shown to be effective in targeting the HIV-1 TAR RNA region of the HIV virus. TAR (Transactivation Response) RNA region, a 59 base pair stem loop structure located at the 5'-end of all nascent viral transcripts interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Ethidium bromide displacement and FRET competition assays have revealed nanomolar binding affinity between neomycin dimers and wildtype TAR RNA while in case of neomycin, only a weak binding was detected. Here, NMR and FID-based comparisons reveal an extended binding interface for neomycin dimers involving the upper stem of the TAR RNA thereby offering an explanation for increased affinities. To further explore the potential of these modified aminosugars we have extended binding studies to include four TAR RNA mutants that display conformational differences with minimal sequence variation. The differences in binding between neomycin and neomycin dimers is characterized with TAR RNA mutants that include mutations to the bulge region, hairpin region, and both the bulge and hairpin regions. Our results demonstrate the effect of these mutations on neomycin binding and our results show that linker functionalities between dimeric units of neomycin can distinguish between the conformational differences of mutant TAR RNA structures.

The effect of the hydrophobic environment on the retro-aldol reaction: comparison to a computationally-designed enzyme.

Recent work on a computationally-designed retroaldolase RA-61 suggested that most of the rate-acceleration brought about by this enzyme was due to non-specific interactions with the aromatic substrate. To provide a benchmark for the role of non-specific interactions in this system, we measured the second-order rate constant for the amine-catalysed retro-aldol reaction of methodol in the presence of non-specific hydrophobic pockets such as micelles. We found that a simple micellar system, that consists of a positively-charged surfactant and a long-chain amine, can accelerate the retro-aldol reaction of methodol by 9500-fold. This effect rivals the 10(5)-fold rate acceleration of RA-61. Similar results were obtained with BSA used as the catalyst, implying that the retro-aldol reaction of methodol can be greatly accelerated by non-specific hydrophobic pockets that contain an amino group.

The arginine-rich RNA-binding motif of HIV-1 Rev is intrinsically disordered and folds upon RRE binding.

Arginine-rich motifs (ARMs) capable of binding diverse RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. The regulatory HIV-1 protein Rev is essential for viral replication and belongs to the ARM family of RNA-binding proteins. During the early stages of the HIV-1 life cycle, incompletely spliced and full-length viral mRNAs are very inefficiently recognized by the splicing machinery of the host cell and are subject to degradation in the cell nucleus. These transcripts harbor the Rev Response Element (RRE), which orchestrates the interaction with the Rev ARM and the successive Rev-dependent mRNA export pathway. Based on established criteria for predicting intrinsic disorder, such as hydropathy, combined with significant net charge, the very basic primary sequences of ARMs are expected to adopt coil-like structures. Thus, we initiated this study to investigate the conformational changes of the Rev ARM associated with RNA binding. We used multidimensional NMR and circular dichroism spectroscopy to monitor the observed structural transitions, and described the conformational landscapes using statistical ensemble and molecular-dynamics simulations. The combined spectroscopic and simulated results imply that the Rev ARM is intrinsically disordered not only as an isolated peptide but also when it is embedded into an oligomerization-deficient Rev mutant. RRE recognition triggers a crucial coil-to-helix transition employing an induced-fit mechanism.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

Backbone ¹HN, ¹³C, and ¹5N resonance assignments of the tandem RNA-binding domains of human DGCR8.

Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493-720 revealed that each dsRBD adopts the canonical αßßßα fold. However, several residues located in important flexible regions including the ß1-ß2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8(core) (residues 493-706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1.

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35-40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all (1)H, (13)C, and (15)N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.

Structures of HIV TAR RNA-ligand complexes reveal higher binding stoichiometries.

Target TAR by NMR: Tripeptides containing arginines as terminal residues and non-natural amino acids as central residues are good leads for drug design to target the HIV trans-activation response element (TAR). The structural characterization of the RNA-ligand complex by NMR spectroscopy reveals two specific binding sites that are located at bulge residue U23 and around the pyrimidine-stretch U40-C41-U42 directly adjacent to the bulge.

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev.

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins' application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.

RNA structure determination by NMR.

This chapter reviews the methodologies for RNA structure determination by liquid-state nuclear magnetic resonance (NMR). The routine production of milligram quantities of isotopically labeled RNA remains critical to the success of NMR-based structure studies. The standard method for the preparation of isotopically labeled RNA for structural studies in solution is in vitro transcription from DNA oligonucleotide templates using T7 RNA polymerase and unlabeled or isotopically labeled nucleotide triphosphates (NTPs). The purification of the desired RNA can be performed by either denaturing polyacrylamide gel electrophoresis (PAGE) or anion-exchange chromatography. Our basic strategy for studying RNA in solution by NMR is outlined. The topics covered include RNA resonance assignment, restraint collection, and the structure calculation process. Selected examples of NMR spectra are given for a correctly folded 30 nucleotide-containing RNA.

Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes.

The translation of the unspliced and partially spliced viral mRNAs that encode the late, structural proteins of HIV-1 depends on the viral-protein Rev. Oligomeric binding of Rev to the Rev response element (RRE) in these mRNAs promotes their export from the nucleus and thus controls their expression. Here, we compared the effects of hydrophobic to hydrophilic mutations within the oligomerization domain of Rev using assays for oligomeric RNA binding, protein structure, and export from the nucleus. Oligomeric RNA binding alone does not correlate well with RNA transport activity in the subset of mutants. However, protein structure as judged by CD spectroscopy does correlate well with Rev function. The oligomeric assembly of Rev-L18T is impaired but exhibits minor defects in structure and retains a basal level of activity in vivo. The prevalence of L18T in infected individuals suggests a positive selection mechanism for L18T modulation of Rev activity that may delay the onset of AIDS.

NMR assignments of HIV-2 TAR RNA.

We report nearly complete assignment for all (1)H, (13)C, (31)P, and (15)N resonances in the 30-nucleotide stem-loop HIV-2 TAR RNA located at the 5' end of all viral mRNAs.

Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure.

Enzymatic synthesis methods for the fluorinated 5'-triphosphate analogues 5F-UTP and 5F-CTP have been developed to facilitate 19F-labeling of RNAs for biophysical studies. HIV-2 TAR RNAs were synthesized using these analogues by in vitro transcription reactions using T7 RNA polymerase. The uniform incorporation of 5F-U or 5F-C analogues into HIV-2 TAR RNA transcripts does not significantly alter the RNA structure or thermodynamic stability. Fluorine observed homonuclear 19F-19F and heteronuclear 19F-1H NOE experiments providing selective distance information are presented and discussed. The availability of efficient synthesis of 5F-UTP, and for the first time, 5F-CTP, will facilitate the use of 5F-labeled RNAs in structural, ligand binding, and dynamic studies of RNAs using the advantages of 19F-labeling.