mRNA structure regulates protein expression through changes in functional half-life.

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.

MicroRNAs Enable mRNA Therapeutics to Selectively Program Cancer Cells to Self-Destruct.

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.

Analysis of RNA nearest neighbor parameters reveals interdependencies and quantifies the uncertainty in RNA secondary structure prediction.

RNA secondary structure prediction is often used to develop hypotheses about structure-function relationships for newly discovered RNA sequences, to identify unknown functional RNAs, and to design sequences. Secondary structure prediction methods typically use a thermodynamic model that estimates the free energy change of possible structures based on a set of nearest neighbor parameters. These parameters were derived from optical melting experiments of small model oligonucleotides. This work aims to better understand the precision of structure prediction. Here, the experimental errors in optical melting experiments were propagated to errors in the derived nearest neighbor parameter values and then to errors in RNA secondary structure prediction. To perform this analysis, the optical melting experimental values were systematically perturbed within the estimates of experimental error and alternative sets of nearest neighbor parameters were then derived from these error-bounded values. Secondary structure predictions using either the perturbed or reference parameter sets were then compared. This work demonstrated that the precision of RNA secondary structure prediction is more robust than suggested by previous work based on perturbation of the nearest neighbor parameters. This robustness is due to correlations between parameters. Additionally, this work identified weaknesses in the parameter derivation that makes accurate assessment of parameter uncertainty difficult. Considerations for experimental design are provided to mitigate these weaknesses are provided.

IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance.

Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3' untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3' UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217 G allele mRNA forms well-defined 3' UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure.

Functionally conserved architecture of hepatitis C virus RNA genomes.

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 high-throughput, 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 cis-acting 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.

The genetic code as expressed through relationships between mRNA structure and protein function.

Structured RNA elements within messenger RNA often direct or modulate the cellular production of active proteins. As reviewed here, RNA structures have been discovered that govern nearly every step in protein production: mRNA production and stability; translation initiation, elongation, and termination; protein folding; and cellular localization. Regulatory RNA elements are common within RNAs from every domain of life. This growing body of RNA-mediated mechanisms continues to reveal new ways in which mRNA structure regulates translation. We integrate examples from several different classes of RNA structure-mediated regulation to present a global perspective that suggests that the secondary and tertiary structure of RNA ultimately constitutes an additional level of the genetic code that both guides and regulates protein biosynthesis.

Exploring RNA structural codes with SHAPE chemistry.

RNA 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 structure-function 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 RNA-protein 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.

TIA nuclear proteins regulate the alternate splicing of lysyl hydroxylase 2.

Synthesis of collagen, a major component of the extracellular matrix, is increased dramatically in fibrotic conditions such as scleroderma. This overaccumulation of collagen is associated with increased pyridinoline cross-links. These cross-links are derived by the action of the alternatively spliced long form of lysyl hydroxylase 2 (LH2), a collagen telopeptide LH. As LH2 (long) is reported to be overexpressed in scleroderma fibroblasts, the regulation of LH2 splicing suggests an important step in controlling fibrosis. Using an LH2 minigene, we have compared the regulation of the alternative splicing pattern of LH2, both endogenously and in the minigene, by the RNA-binding splicing proteins TIA-1 and TIAL1 (T-cell-restricted intracellular antigens). A decrease in the ratio of LH2 (long) to LH2 (short) was observed in fibroblasts from TIAL1 knockout mice, and in HEK293 cells knocked down for TIA-1 and TIAL1. As a corollary, overexpression of TIA-1/TIAL1 in HEK293 cells resulted in an increase in LH2 (long) minigene transcripts, accompanied by a decrease in LH2 (short). In scleroderma fibroblasts, a double TIA-1/TIAL1 knockdown reduced the ratio of LH2 (long) to LH2 (short) by over fivefold compared to controls. Identification of these TIA regulatory factors therefore suggests a tool to manipulate cellular LH2 levels in scleroderma so that potential intervention therapies may be identified.

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc.

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.

A Requirement for the Saccharomyces cerevisiae Paf1 complex in snoRNA 3' end formation.

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.