Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform

Although phosphorylation directs serine-arginine (SR) proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of protein phosphatase 1 (PP1) with the SR protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. Disruption of RRM1-PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR protein-phosphatase platform balances phosphorylation levels in a "goldilocks" region for the proper subnuclear storage of an SR protein splicing factor

Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets

Cigarette Smoke Triggers IL-33–associated Inflammation in a Model of Late-Stage Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a worldwide threat. Cigarette smoke (CS) exposure causes cardiopulmonary disease and COPD and increases the risk for pulmonary tumors. In addition to poor lung function, patients with COPD are susceptible to bouts of dangerous inflammation triggered by pollutants or infection. These severe inflammatory episodes can lead to additional exacerbations, hospitalization, further deterioration of lung function, and reduced survival. Suitable models of the inflammatory conditions associated with CS, which potentiate the downward spiral in patients with COPD, are lacking, and the underlying mechanisms that trigger exacerbations are not well understood. Although initial CS exposure activates a protective role for vascular endothelial growth factor (VEGF) functions in barrier integrity, chronic exposure depletes the pulmonary VEGF guard function in severe COPD. Thus, we hypothesized that mice with compromised VEGF production and challenged with CS would trigger human-like severe inflammatory progression of COPD. In this model, we discovered that CS exposure promotes an amplified IL-33 cytokine response and severe disease progression. Our VEGF-knockout model combined with CS recapitulates severe COPD with an influx of IL-33-expressing macrophages and neutrophils. Normally, IL-33 is quickly inactivated by a post-translational disulfide bond formation. Our results reveal that BAL fluid from the CS-exposed, VEGF-deficient cohort promotes a significantly prolonged lifetime of active proinflammatory IL-33. Taken together, our data demonstrate that with the loss of a VEGF-mediated protective barrier, the CS response switches from a localized danger to an uncontrolled long-term and long-range, amplified, IL-33-mediated inflammatory response that ultimately destroys lung function

The formation of individual contacts represented for each individual trefoil in Interleukin-33.

<p>Plots of q versus Q_CA for interactions between individual strands or within trefoil units are represented. Each plot represents the formation of contacts for the given strands (q) versus the formation of all native contacts within IL-33 (Q_CA). The plots for contacts located in Trefoil 1 are highlighted in cyan, Trefoil 2 in blue, and Trefoil 3 in green. The contacts formed between trefoils are highlighted in black. The dashed lines compare the overall foldedness of the protein.</p

Experimental kinetic and equilibrium folding data for Interleukin-33.

<p>(A) The chevron plot of the observed rate of folding and unfolding as a function of final denaturant concentration for IL-33 (upper panel) and the equilibrium titrations as a function of final GdmCl concentration (lower panel). (B) The chevron plot for IL-33 (upper panel) and the equilibrium unfolding curve for IL-33 as a function of final urea concentration (lower panel) constructed as in (A). Representative exponential fits of folding for GdmCl and Urea are shown as inset panels within the chevron plots. Grey triangles within the chevron plots represent the second fitted phase in strongly native conditions. The data are well fit by a single exponential over most denaturant concentrations except in strongly native conditions (<1 M GdmCl and <2 M urea, respectively) where a rollover appears and the refolding rates are fit to a double exponential equation. The total chevron plots are fit to a two-state equation over the linear regime. The equilibrium curves are fitted with a two-state equation.</p

Equilibrium Titrations.

<p>Data for Urea and GdmCl were both fit with a two state equation.</p><p>Errors are reported as standard deviation.</p><p>Equilibrium Titrations.</p

Free energy profile of Interleukin-33 and its individual trefoils determined from structure-based simulations.

<p>(A) A plot of the free energy as a function of Q_CA. The denatured basin is populated at a Q_CA of 0.2, the native basin is populated at a Q_CA of 0.9 and the transition region has two transition states (TS) at Q_CA of 0.4 (TS₁) and at Q_CA of 0.7 (TS₂). The shaded region highlights the transition state region for IL-33. Representative structures of the species present at each of the two transition states are shown above the profile plot. (B) Two dimensional free-energy landscapes as a function of q and Q_CA for Trefoils 1, 2 and 3. The occupancy of states is represented by a color scale where red represents lowest occupancy and blue represents highest occupancy.</p

Kinetic Data.

<p>Data for Urea and GdmCl were both fit at ± 2M of the midpoint.</p><p>Errors are reported as standard deviation.</p><p>Kinetic Data.</p