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

Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA

<div><p>The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from <i>Schizosaccharomyces pombe</i> at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in <i>Saccharomyces cerevisiae</i> shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in <i>S</i>. <i>cerevisiae</i>, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.</p></div

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

Statistics of data collection and structural refinement.

<p>Statistics of data collection and structural refinement.</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

Structure of <i>S</i>. <i>pombe</i> Utp24.

<p>(A) Ribbon representation of spUtp24 structure. The secondary structures and the N- and C-termini are labeled. The catalytic residues and Zn-coordinating residues are shown as sticks and the bound Zn ion as sphere. (B) Structure of the Zn-finger motif. (C) Structural alignment of Utp24 and Utp23 (PDB code 4MJ7). (D) Alignment of crystal structure of spUtp24 with cryo-EM structures of scUtp24 (PDB code 5WLC) and ctUtp24 (PDB code 5OQL). (E) Multiple sequence alignment of Utp24. The Utp24 sequences from <i>S</i>. <i>cerevisiae</i> (Sc), <i>Homo sapiens</i> (Hs), <i>Gallus gallus</i> (Gg), <i>Drosophila melanogaster</i> (Dm), <i>Arabidopsis thaliana</i> (At) and <i>S</i>. <i>pombe</i> (Sp) are aligned. Residues conserved in at least 100%, 80% and 60% of these sequences are shaded in black, gray and light gray, respectively. The secondary structure elements observed in the crystal structure are shown on the top. The dotted line denotes the region not resolved in crystal structure. At the bottom of the alignment, the catalytic and Zn-coordinating residues are denoted by triangles and circles, respectively. A vertical bar marks the joining point of scUtp24 and hUTP24 in the chimera protein.</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

snR30 is required for the stable association of Rrp7 to preribosome.

<p>(A) Depletion of Rrp7–HTP in yeast <i>GAL::RRP7–HTP</i> after shift to glucose medium. Rrp7–HTP was detected by Western blotting using PAP. Equal amounts of total protein were loaded. (B–C) Sedimentation behavior of snR30 in the presence (B) and absence (C) of Rrp7. Extracts of <i>GAL::RRP7–HTP</i> cells grown in galactose (Gal) or glucose (Glu) medium for 16 h were fractionated on 7%–50% sucrose gradients. The distributions of snR30, snR10, and U3 were analyzed by Northern blotting. The polyribosome profiles are displayed. (D) Depletion of snR30 in yeast <i>GAL::SNR30</i>/<i>RRP7–HTP</i> after shift to glucose medium. snR30 was detected by Northern blotting. Equal amounts of total RNA (1 µg) were loaded. (E–F) Sedimentation behavior of Rrp7 in the presence (E) and absence (F) of snR30. Extracts of <i>GAL::SNR30/RRP7–HTP</i> cells grown in galactose or glucose medium for 14 h were fractionated on 7%–50% sucrose gradients. The distributions of Rrp7, snR10, and U3 were analyzed. (G) Schematic structure and cleavage sites of 35S pre-rRNA. (H) Association of Rrp7 with preribosomes. Yeast cells <i>BY4741</i>, <i>RRP7–HTP</i>, and <i>GAL::SNR30/RRP7–HTP</i> were grown in galactose or glucose medium for 14 h. Total cell lysates (TCLs) and immunoprecipitations (IP) of IgG Sepharose were analyzed by Western blotting to detect Rrp7–HTP and by Northern blotting to detect copurifed U3 snoRNA and pre-rRNAs. A probe D-A2 that hybridizes to a region between sites D and A2 was used to detect 35S, 23S, and 20S pre-rRNAs. The minor fast-migrating band of Rrp7–HTP marked by asterisk might be degradation or modification products and its identity was not studied.</p

NMR structure and dynamics of MSL2 CXC domain.

<p>(A) Structural superposition of the 20 lowest energy NMR structures. The Cα traces of residues 521–566 and three zinc ions (spheres) are shown in cross-eye stereoview. (B) Steady-state ¹H-¹⁵N heteronuclear NOE values are plotted as a function of residue number. The experiment was conducted for CXC-2, which contains residues 517–572 plus three extra N-terminal residues from the vector. No data were obtained for proline residues that lack amide proton. Error bars represent the experimental uncertainties estimated from the spectrum background noise.</p

Refinement statistics for the 20 lowest energy NMR structures of MSL2 CXC domain.

<p>Refinement statistics for the 20 lowest energy NMR structures of MSL2 CXC domain.</p