SUMMARY RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3′ UTR binding sites were more highly stabilized than transcripts with only 3′ UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

The ability to predict the impact of cis-regulatory sequence on gene expression would facilitate discovery in fundamental and applied biology. Here, we combine polysome profiling of a library of 280,000 randomized 5′ UTRs with deep learning to build a predictive model that relates human 5′ UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5′ UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,000 truncated human 5′ UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 SNVs associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease. The sequence of the 5′ untranslated region (5′ UTR) is a primary determinant of translation efficiency 1,2. While many cis-regulatory elements within human 5′ UTRs have been characterized individually, the field still lacks a means to accurately predict protein expression from 5′ UTR sequence alone, limiting the ability to estimate the effects of genome-encoded variants and the ability to engineer 5′ UTRs for precise translation control. Massively parallel reporter assays (MPRAs)-methods that assess thousands to millions of sequence variants in a single experiment-coupled with machine learning have proven † Corresponding author. gseelig@uw.edu. Author contributions P.J.S and B.W. designed and performed experiments, performed data analysis and modeling, and wrote the manuscript. D.R. performed fluorescence validation experiments. V.P. and I.M. wrote the manuscript. D.R.M. helped design polysome profiling. G.S. designed experiments and wrote the manuscript.

Background: There are few data in asthma relating airway physiology, inflammation and remodelling and the relative effects of inhaled corticosteroid (ICS) treatment on these parameters. A study of the relationships between spirometric indices, airway inflammation, airway remodelling, and bronchial hyperreactivity (BHR) before and after treatment with high dose inhaled fluticasone propionate (FP 750 µg bd) was performed in a group of patients with relatively mild but symptomatic asthma. Methods: A double blind, randomised, placebo controlled, parallel group study of inhaled FP was performed in 35 asthmatic patients. Bronchoalveolar lavage (BAL) and airway biopsy studies were carried out at baseline and after 3 and 12 months of treatment. Twenty two normal healthy non-asthmatic subjects acted as controls. Results: BAL fluid eosinophils, mast cells, and epithelial cells were significantly higher in asthmatic patients than in controls at baseline (p<0.01). Subepithelial reticular basement membrane (rbm) thickness was variable, but overall was increased in asthmatic patients compared with controls (p<0.01). Multiple regression analysis explained 40% of the variability in BHR, 21% related to rbm thickness, 11% to BAL epithelial cells, and 8% to BAL eosinophils. The longitudinal data corroborated the cross sectional model. Forced expiratory volume in 1 second improved after 3 months of treatment with FP with no further improvement at 12 months. PD 20 improved throughout the study. BAL inflammatory cells decreased following 3 months of treatment with no further improvement at 12 months (p<0.05 v placebo). Rbm thickness decreased in the FP group, but only after 12 months of treatment (mean change -1.9, 95% CI -3 to -0.7 µm; p<0.01 v baseline, p<0.05 v placebo). A third of the improvement in BHR with FP was associated with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodelling. Conclusion: Physiology, airway inflammation and remodelling in asthma are interrelated and improve with ICS. Changes are not temporally concordant, with prolonged treatment necessary for maximal benefit in remodelling and PD 20 . Determining the appropriate dose of inhaled steroids only by reference to symptoms and lung function, as specified in current international guidelines, and even against indices of inflammation may be over simplistic. The results of this study support the need for early and long term intervention with ICS, even in patients with relatively mild asthma.

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.