The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.

Cardiac resynchronization therapy in patients with chronic heart failure is associated with anti-inflammatory and anti-remodeling effects.

OBJECTIVES: Proinflammatory cytokines, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in left ventricular (LV) structural remodeling. We aimed to investigate the effects of cardiac resynchronization therapy (CRT) on serum levels of amino-terminal prohormone B-type natriuretic peptide (NT-proBNP), some interleukins (IL-1ß, IL-6, IL-8), MMP-2 and TIMP-2 in patients with chronic heart failure (CHF). DESIGN AND METHODS: We studied 27 patients (15 M/12 F) with CHF, III-IV NYHA class, implanted with a biventricular pacemaker/defibrillator and 40 healthy subjects (23 M/17 F). Blood samples were collected at baseline and 1 week, 3, 6, and 12 months after CRT device implantation. Cardiac function was assessed echocardiographically. RESULTS: CRT induced significant improvement in the NYHA class (baseline 3.2±0.5 vs. 1.0 at 12 months, P=0.0002) and significant LV reverse remodeling, with a 41% (P=0.001) reduction in LV end-systolic volume (LVESV). This was associated with a significant reduction in serum NT-proBNP, IL-6 and IL-8. Positive extracellular matrix remodeling was illustrated by decreasing levels of MMP-2 and increasing TIMP-2. MMP-2/TIMP-2 ratio decreased with 55% (P=0.003) from baseline value at 12 months and the correlation with LVESV reduction was 0.41 (P=0.001). CONCLUSIONS: Structural response to CRT is associated with reduced immune activation and positive extracellular matrix remodeling.