Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.

BACKGROUND The response to treatment for asthma is characterized by wide interindividual variability, with a significant number of patients who have no response. We hypothesized that a genomewide association study would reveal novel pharmacogenetic determinants of the response to inhaled glucocorticoids. METHODS We analyzed a small number of statistically powerful variants selected on the basis of a family-based screening algorithm from among 534,290 single-nucleotide polymorphisms (SNPs) to determine changes in lung function in response to inhaled glucocorticoids. A significant, replicated association was found, and we characterized its functional effects. RESULTS We identified a significant pharmacogenetic association at SNP rs37972, replicated in four independent populations totaling 935 persons (P = 0.0007), which maps to the glucocorticoid-induced transcript 1 gene (GLCCI1) and is in complete linkage disequilibrium (i.e., perfectly correlated) with rs37973. Both rs37972 and rs37973 are associated with decrements in GLCCI1 expression. In isolated cell systems, the rs37973 variant is associated with significantly decreased luciferase reporter activity. Pooled data from treatment trials indicate reduced lung function in response to inhaled glucocorticoids in subjects with the variant allele (P = 0.0007 for pooled data). Overall, the mean (± SE) increase in forced expiratory volume in 1 second in the treated subjects who were homozygous for the mutant rs37973 allele was only about one third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2 ± 1.6% vs. 9.4 ± 1.1%), and their risk of a poor response was significantly higher (odds ratio, 2.36; 95% confidence interval, 1.27 to 4.41), with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability. CONCLUSIONS A functional GLCCI1 variant is associated with substantial decrements in the response to inhaled glucocorticoids in patients with asthma. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT00000575.)

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

R NA is the central conduit for gene expression. This role depends on an ability to encode information at two levels: in its linear sequence and in the complex structures RNA can form by folding back on itself. Understanding the global structureÀfunction interrelationships mediated by RNA remains a great challenge in molecular and structural biology. In this Account, we discuss evolving work in our laboratory focused on creating facile, generic, quantitative, accurate, and highly informative approaches for understanding RNA structure in biologically important environments.The core innovation derives from our discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl in RNA is gated by local nucleotide flexibility. The 2'-hydroxyl is reactive at conformationally flexible positions but is unreactive at nucleotides constrained by base pairing. Sites of modification in RNA can be detected efficiently either using primer extension or by protection from exoribonucleolytic degradation. This technology is now called SHAPE, for selective 2'-hydroxyl acylation analyzed by primer extension (or protection from exoribonuclease).SHAPE reactivities are largely independent of nucleotide identity but correlate closely with model-free measurements of molecular order. The simple SHAPE reaction is thus a robust, nucleotide-resolution, biophysical measurement of RNA structure. SHAPE can be used to provide an experimental correction to RNA folding algorithms and, in favorable cases, yield kilobase-scale secondary structure predictions with high accuracies.SHAPE chemistry is based on very simple reactive carbonyl centers that can be varied to yield slow-and fast-reacting reagents. Differential SHAPE reactivities can be used to detect specific RNA positions with slow local nucleotide dynamics. These positions, which are often in the C2'-endo conformation, have the potential to function as molecular timers that regulate RNA folding and function. In addition, fast-reacting SHAPE reagents can be used to visualize RNA structural biogenesis and RNAÀprotein assembly reactions in one second snapshots in very straightforward experiments.The application of SHAPE to challenging problems in biology has revealed surprises in well-studied systems. New regions have been identified that are likely to have critical functional roles on the basis of their high levels of RNA structure. For example, SHAPE analysis of large RNAs, such as authentic viral RNA genomes, suggests that RNA structure organizes regulatory motifs and regulates splicing, protein folding, genome recombination, and ribonucleoprotein assembly. SHAPE has also revealed limitations to the hierarchical model for RNA folding. Continued development and application of SHAPE technologies will advance our understanding of the many ways in which the genetic code is expressed through the underlying structure of RNA.

RNA synthesis and processing are coordinated by proteins that associate with RNA polymerase II (pol II) during transcription elongation. The yeast Paf1 complex interacts with RNA pol II and mediates histone modifications during elongation. To elucidate the functions of this complex, we isolated missense mutations in the gene encoding the Rtf1 subunit and used them to identify functionally interacting proteins. We identified NAB3 as a dosage suppressor of rtf1. Nab3, together with Nrd1, directs 3' end formation of nonpolyadenylated RNA pol II transcripts, such as snoRNAs. Deletion of Paf1, but not the Set1, Set2, or Dot1 histone methyltransferases, causes accumulation of snoRNA transcripts that are extended at their 3' ends. The Paf1 complex associates with and facilitates Nrd1 recruitment to the SNR47 gene, suggesting a direct involvement in 3' end formation. Our results reveal a posttranscriptional function for the Paf1 complex, which appears unrelated to its role in histone methylation.

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large group of nuclear RNA binding proteins, several of which regulate mRNA splicing. The protein family of hnRNP H and hnRNP F is one group of hnRNPs that have been found to play important roles in the regulation of alternative splicing decisions (5). hnRNP H and hnRNP F are two closely related proteins that bind to the RNA sequence DGGGD (6). Intriguingly, hnRNP H has been shown to have either enhancing or silencing activity, depending on the context of the binding site. On the one hand, hnRNP H activates exon inclusion by binding G-rich intronic elements downstream of the 5Ј splice site in c-src (15, 29), human immunodeficiency virus type 1 (HIV-1) (7), Bcl-X (22, 34), GRIN1 (23), and myelin (34) transcripts, while on the other hand, it silences exons when bound to exonic elements in ␤-tropomyosin (14, 21), 24), and ␣-tropomyosin (16) transcripts. Recently, Martinez-Contreras et al. proposed that the hnRNP H/F family, as well as the hnRNP A/B family, could stimulate the splicing of long introns in vitro by binding near the ends of these introns and dimerizing, thus looping out the intron (26). The fact that the function of hnRNP H and hnRNP F depends upon context suggests that the poorly understood mechanism by which hnRNP H and hnRNP F regulate exons involves a complex series of RNAprotein and protein-protein interactions.Although the mechanism of splicing regulation is poorly understood, the domains of the hnRNP H and hnRNP F proteins have been well characterized. Figure 1 shows an alignment of the amino acid sequences for hnRNP H and hnRNP F family members: hnRNP H1 (GenBank accession number NP_005511), hnRNP H2 (accession number NP_001027565), hnRNP F (accession number NP_001091674), and the two alternatively spliced isoforms of hnRNP H3 (accession nu...

Background and RationaleAtypical antipsychotics exhibit metabolic side effects including diabetes mellitus and obesity. The adverse events are preceded by acute worsening of oral glucose tolerance (oGTT) along with reduced plasma free fatty acids (FFA) and leptin in animal models. It is unclear whether the same acute effects occur in humans.Methodology/Principal FindingsA double blind, randomized, placebo-controlled crossover trial was conducted to examine the potential metabolic effects of olanzapine in healthy volunteers. Participants included male (8) and female (7) subjects [18–30 years old, BMI 18.5–25]. Subjects received placebo or olanzapine (10 mg/day) for three days prior to oGTT testing. Primary endpoints included measurement of plasma leptin, oral glucose tolerance, and plasma free fatty acids (FFA). Secondary metabolic endpoints included: triglycerides, total cholesterol, high- and low-density lipoprotein cholesterol, heart rate, blood pressure, body weight and BMI. Olanzapine increased glucose Area Under the Curve (AUC) by 42% (2808±474 vs. 3984±444 mg/dl·min; P = 0.0105) during an oGTT. Fasting plasma leptin and triglycerides were elevated 24% (Leptin: 6.8±1.3 vs. 8.4±1.7 ng/ml; P = 0.0203) and 22% (Triglycerides: 88.9±10.1 vs. 108.2±11.6 mg/dl; P = 0.0170), whereas FFA and HDL declined by 32% (FFA: 0.38±0.06 vs. 0.26±0.04 mM; P = 0.0166) and 11% (54.2±4.7 vs. 48.9±4.3 mg/dl; P = 0.0184), respectively after olanzapine. Other measures were unchanged.Conclusions/SignificanceOlanzapine exerts some but not all of the early endocrine/metabolic changes observed in rodent models of the metabolic side effects, and this suggest that antipsychotic effects are not limited to perturbations in glucose metabolism alone. Future prospective clinical studies should focus on identifying which reliable metabolic alterations might be useful as potential screening tools in assessing patient susceptibility to weight gain and diabetes caused by atypical antipsychotics.Trial RegistrationClinicalTrials.gov NCT00741026

The advent of therapeutic mRNAs significantly increases the possibilities of protein-based biologics beyond those that can be synthesized by recombinant technologies (eg, monoclonal antibodies, extracellular enzymes, and cytokines). In addition to their application in the areas of vaccine development, immune-oncology, and protein replacement therapies, one exciting possibility is to use therapeutic mRNAs to program undesired, diseased cells to synthesize a toxic intracellular protein, causing cells to self-destruct. For this approach to work, however, methods are needed to limit toxic protein expression to the intended cell type. Here, we show that inclusion of microRNA target sites in therapeutic mRNAs encoding apoptotic proteins, Caspase or PUMA, can prevent their expression in healthy hepatocytes while triggering apoptosis in hepatocellular carcinoma cells.