Journal: PLoS Biology

Article Title: The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

doi: 10.1371/journal.pbio.1002083

Figure Lengend Snippet: (A) Cultures of AJY3711 ( P GAL - 3xHA-DHR1 ) expressing untagged WT Dhr1 (pAJ3082), WT Dhr1–13myc (pAJ2311), or dhr1 K420A - 13myc (pAJ3081) were shifted to glucose media for 6 h to deplete 3xHA-Dhr1. RNA was prepared from whole cell extracts (Input) or immunoprecipitated samples (IP) and separated by electrophoresis through agarose/formaldehyde gels or denaturing polyacrylamide gels for the A0-A1 fragment and U3. RNAs were detected by Northern blotting using probes specific to A2-A3 (AJO603), D-A2 (AJO130), A0-A1 (AJO1850), and U3 (AJO1686). (B) Table of proteins identified by MS in the Dhr1 K420A particle. Only proteins with at least three peptide-spectrum matches are listed. (C) The CPK (red), 18S rRNA (cyan), and r-proteins identified in the Dhr1 K420A particle (3B) are shown in orange in the structure of the mature S . cerevisiae 40S subunit (left). For comparison the proteins missing from the Dhr1 K420A particle are shown in yellow on the right. The Dhr1 K420A particle likely adopts a more open conformation in the absence of r-proteins.

Article Snippet: Resulting spectra were searched against the UniProtKB YEAST Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FASTA using Sequest HT in the Proteome Discoverer v1.4 software (Thermo Scientific).

Techniques: Expressing, Immunoprecipitation, Electrophoresis, Northern Blot, Comparison

Journal: PLoS Biology

Article Title: The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

doi: 10.1371/journal.pbio.1002083

Figure Lengend Snippet: (A) The parental and a Dhr1-HTP tagged strain were subjected to the CRAC protocol (see ), cross-linked RNA was partially digested, radioactively labeled, ligated to linkers, and after nickel purification resolved on a 4%–12% NuPAGE gel. Protein-RNA complex was transferred to nitrocellulose and RNA was extracted from the regions indicated by a red dashed box. (B) Dhr1 preferentially cross-links to U3. Reads from Dhr1 ( n = 2) and a negative control CRAC experiment were mapped to the 2008 S . cerevisiae genomic reference sequence and mapped reads were assigned to genomic features. The histogram shows the average percentage of all mapped reads that contained box C/D and (C) box H/ACA snoRNA sequences. Note that only a very small fraction of the reads from the control experiment mapped to snoRNAs. (D) Dhr1 preferentially cross-links to the 5′ end of the U3. Plotted is the average read distribution frequency over the U3A (snR17A) gene generated from two Dhr1 CRAC datasets. A schematic representation of the U3 gene and functional sequence elements are indicated below the plot. (E) Same as in (D) but for nucleotide substitutions. The secondary structure of the U3 was adopted from Granneman and colleagues and generated using VARNA ( http://varna.lri.fr ). The coloring indicates the frequency by which the nucleotide was substituted. Additional supporting data are provided in .

Article Snippet: Resulting spectra were searched against the UniProtKB YEAST Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FASTA using Sequest HT in the Proteome Discoverer v1.4 software (Thermo Scientific).

Techniques: Labeling, Purification, Negative Control, Sequencing, Control, Generated, Functional Assay

Journal: Nature Communications

Article Title: An inhibitory segment within G-patch activators tunes Prp43-ATPase activity during ribosome assembly

doi: 10.1038/s41467-024-54584-5

Figure Lengend Snippet: a Left panel: Prp43-TAP eluate was separated on a NuPAGE 4–12% Bis-Tris gradient gel and analysed by Silver staining or Western blotting using α-CBP antibody. WT (BY4741 untagged strain) was used as negative control. Middle panel: Prp43-TAP proteome was identified by label-free quantitative mass spectrometry analysis. The data was analysed with the R package prolfqua . The plot shows a Bayesian false discovery rate (BFDR) of less than 10% as a function of empirical fold change (EFC) score >2. Right panel: Statistically significant proteins ( p -value < 0.001) were grouped based on the DAVID functional GO clustering analysis. This experiment was performed independently three times with similar results. b Sequence alignments of Tma23 with known G-patch proteins across species: (Sc) Saccharomyces cerevisiae ; (Sp) Schizosaccharomyces pombe ; (Dm) Drosophila melanogaster ; (Dr) Danio rerio ; (Hs) Homo sapiens . Brace-helix and brace-loop as defined previously are indicated on top. The alignments were made with MAFFT and visualized with Jalview (version 2.11.2.5) – . Asterisks indicate residues substituted for functional studies. c , Tma23-TAP and Pxr1-TAP eluates were separated on a NuPAGE 4-12% Bis-Tris gradient gel and subjected to Western blotting using antibodies directed against Tma23, Pxr1, and Prp43. This experiment was performed independently three times with similar results. Source data are provided as a Source Data file.

Article Snippet: In short, MS2 spectra of a minimum of 2 peptides per protein were searched against the Saccharomyces cerevisiae (strain ATCC 204508/S288c) UniProtKB reference proteome (UP000002311, retrieved on 2022-09-05) by the MSFragger search engine 3.4, allowing for one missed tryptic cleavage and fixed carbamidomethylation of Cysteine, variable Methionine oxidation, and variable acetylation of the protein N-terminus after Methionine removal.

Techniques: Silver Staining, Western Blot, Negative Control, Mass Spectrometry, Functional Assay, Sequencing