Accuracy of wind observations from open-ocean buoys: Correction for flow distortion

The comparison of equivalent neutral winds obtained from (a) four WHOI buoys in the subtropics and (b) scatterometer estimates at those locations reveals a root-mean-square (RMS) difference of 0.56-0.76 m/s. To investigate this RMS difference, different buoy wind error sources were examined. These buoys are particularly well suited to examine two important sources of buoy wind errors because: (1) redundant anemometers and a comparison with numerical flow simulations allow us to quantitatively assess flow distortion errors, and (2) one-minute sampling at the buoys allows us to examine the sensitivity of buoy temporal sampling/averaging in the buoy-scatterometer comparisons. The inter-anemometer difference varies as a function of wind direction relative to the buoy wind vane and is consistent with the effects of flow distortion expected based on numerical flow simulations. Comparison between the anemometers and scatterometer winds supports the interpretation that the inter-anemometer disagreement, which can be up to 5% of the wind speed, is due to flow distortion. These insights motivate an empirical correction to the individual anemometer records and subsequent comparison with scatterometer estimates show good agreement

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs

Development of an RNA–protein crosslinker to capture protein interactions with diverse RNA structures in cells

Characterization of RNA–protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA–protein crosslinker (AMT–NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT–NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA–protein interactions in cells

Functional assay of Utp24 in <i>S</i>. <i>cerevisiae</i>.

<p>(A) Depletion of Utp24 in the Utp24 shuffle strain after transfer to glucose medium. Total proteins were resolved with SDS-PAGE and analyzed with Western blot using anti-HA and PAP antibodies. The PAP antibody recognizes protein A in the TAP-tag. (B) Growth assay. The utp24Δ/ENP1-TAP strain complemented by a <i>URA3</i> plasmid expressing wild-type Utp24 under control of a <i>GAL</i> promoter was transformed with a <i>LEU2</i> plasmid expressing WT or mutant Utp24, Utp24 from human or <i>S</i>. <i>pombe</i>, or a chimera protein containing the N-terminal residues 1–60 (N) of scUtp24 and the PIN domain (residues 65–198) of hUTP24. Five folds serial dilutions of cells were spotted on Ura- and Leu-deficient SC medium containing galactose (Gal) or glucose (Glu) and incubated at 30°C. (C) Association of Utp24 mutants with 90S. The strains described in (B) were grown in glucose for 14 h. Total cell lysates (TCL) and immunoprecipitations (IP) of IgG coated beads were analyzed with Western blot using PAP, anti-Flag and anti-Krr1 antibodies. Positions of molecular weight makers are indicated. (D) An agarose gel showing rRNA stained by ethidium bromide. Total RNAs were extracted from ENP1-TAP strain, Utp24 shuffle strain grown in YPG and then in YPD, or Utp24 shuffle strain expressing the D138N mutant of Utp24 grown in YPD. (E) Ribosome profile. Extracts of Utp24 shuffle strains transformed with a plasmid expressed wild-type or D138N mutant Utp24 or an empty plasmid grown in glucose were fractionated on 7%-50% sucrose gradients.</p

Heatmap of AFs in pre-ribosomes.

<p>Utp24 shuffle stains alone or expressing WT or mutant Utp24 were grown in YPG and shifted to YPD for 14 h to deplete wild type Utp24. The first two samples were previously reported and included for comparison [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195723#pone.0195723.ref006" target="_blank">6</a>]. The first sample (ITS1-239/Noc4-TAP) was purified via a plasmid-derived pre-18S fragment ending at position 293 of ITS1 and Noc4-TAP and stands for a fully assembled 90S particle that lacks labile AFs and contains an unprocessed 5' ETS. The second Noc4-TAP sample was purified in two steps. All other samples were purified with single step of IgG immunoprecipitation. Proteins are color-coded according to their relative spectral abundance factor (RSAF) values relative to the UTPB proteins. Proteins in magenta are labile AFs diminished in fully assembled 90S particles.</p

Northern blot analysis of pre-rRNA processing intermediates.

<p>Utp24 shuffle strains transformed with a plasmid encoding wild-type or variant Utp24 were grown YPG and then shifted to YPD for 14 h. RNAs extracted from total cell lysate (TCL) and IgG immunoprecipitations (IP) were resolved in 1.2% agarose-formaldehyde gels and strained by ethidium bromide (EB). RNAs were transferred to Hybond N⁺ membranes, hybridized against ³²P-labeled probes and visualized by autoradiography. Asterisk and double asterisk denote non-specific bands of 25S and 18S rRNA, respectively.</p