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RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.non-coding RNA | RNA processing | miRNA | mass spectrometry R NA molecules function together with specific binding proteins to regulate cellular pathways at the levels of transcription, posttranscriptional modification, and translation (1-4). For example, microRNAs (miRNA), siRNAs, and piwi-interacting RNAs (piRNAs) regulate more than 30% of mammalian gene expression (5). Small nucleolar RNAs (snoRNAs) govern the sites and efficiencies of RNA chemical modifications in cells (6), whereas long noncoding RNAs (lncRNAs), such as HOTAIR and MALAT1, have been implicated in chromatin remodeling, transcriptional activation, and tumorigenesis (7,8). UTRs of mRNA transcripts are also known to interact with regulatory proteins to control their expression level as well as their stability. Understanding how these RNAs function in cells and how they may be manipulated for therapeutic purposes is an important goal that spans many areas of biology.Although new ncRNAs are being discovered at a rapid pace, determination of their biochemical activities is often slow. A major barrier to such functional analysis is the current difficulty of identifying RNA-binding partners that associate with specific transcripts and participate in their biological behavior. Despite the development of various RNA affinity purification methods, the expense and/or technical challenges associated with each approach has precluded its use by nonspecialists or in high-throughput discovery experiments. Current strategies for identifying RNA-binding proteins that associate with specific transcripts involve the use of affinity tags, including biotin, aptamers, and particular proteinbinding sequences (9-13). In each case, however, the modest affinity or specificity of tag recognition and the difficulty of selective elution complicate sample analysis. A strategy that will allow simple and rapid identification of proteins that associate selectively with particu...

OBJECTIVE:To comprehensively evaluate clinical, economic, and patient-reported outcomes associated with various therapeutic classes of asthma controller medications. PATIENTS AND METHODS:This observational study, which used administrative claims data from US commercial health plans, included patients with asthma aged 18 through 64 years who filled a prescription for at least 1 asthma controller medication from September 1, 2003, through August 31, 2005. Outcome metrics included the use of short-acting b-agonists (SABAs), the use of oral corticosteroids, inpatient (INP)/emergency department (ED) visits, and asthma-related health care costs. A subset of 5000 patients was randomly selected for a survey using the Mini-Asthma Quality of Life Questionnaire, the Work Productivity and Activity Impairment questionnaire, and the Asthma Therapy Assessment Questionnaire.RESULTS: Of 56,168 eligible patients, 823 returned completed questionnaires. Compared with inhaled corticosteroids (ICSs), leukotriene modifiers (LMs) were associated with lower odds of INP/ED visits (odds ratio [OR], 0.80; P<.001), lower odds of using 6 or more SABA canisters (OR, 0.81; P<.001), and higher annual cost ($193; P<.001). In the subgroup analysis of adherent patients, LMs were associated with higher odds of INP/ED visits (OR, 1.74; P=.04), lower odds of using 6 or more SABA canisters (OR, 0.46; P<.001), and higher annual cost ($235; P<.001). Inhaled corticosteroids and LMs had a comparable impact on all patient-reported outcomes. For combination therapy, ICS plus a long-acting b-agonist consistently showed at least equivalent or better outcomes in the use of SABAs and oral corticosteroids, the risk of INP/ED visits, cost, asthma control level, quality of life, and impairment in productivity and activity.CONCLUSION: Inhaled corticosteroids were associated with a lower risk of INP/ED visits, and a lower cost if adherence was achieved. When adherence cannot be achieved, LMs may be a reasonable alternative. Combination therapy with ICS plus a longacting b-agonist was associated with better or equivalent clinical, economic, and patient-reported outcomes.

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