Journal: Nature Communications

Article Title: The structural basis of N -acyl-α-amino-β-lactone formation catalyzed by a nonribosomal peptide synthetase

doi: 10.1038/s41467-019-11383-7

Figure Lengend Snippet: Structure of the BD-ObiF1 module. a ObiF1 from Burkholderia diffusa possesses a C-A-PCP-TE-MLP domain architecture. b Front and back side view of the holo-ObiF1 module. c The pPant arm is positioned in the C domain near the canonical catalytic C domain His residue (His144). Electron density (contoured at 3.0σ) calculated with mF o –DF c coefficients from an omit map (ligand occupancy set at zero) generated with simulated annealing. d A surface representation of the A and C domain interface illustrates the salt-bridging interactions that occur between the N-terminal region of the MLP domain and the A domain

Article Snippet: Genes encoding ObiF2 (WP_059467197.1), ObiH (WP_059467200.1), and ObiD (WP_059467195.1), were synthesized, codon optimized (Supplementary Figs. – ), and cloned into a pET28 vector (NdeI/HindIII) encoding for an N -terminal hexahistidine tag (Genscript; Piscataway, NJ).

Techniques: Residue, Generated

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: iRAP and iRAP-identified Hel2 crosslink sites, sequence and structure elements and conservation. a Schematic representation of the iRAP procedure. b Positioning of sequence and structural elements as well as crosslinked amino acids revealed by iRAP within the Hel2 primary structure. See Supplementary Fig. for a structure prediction of Hel2. c Multiple sequence alignment of crosslink-proximal amino acids of Hel2 from S. cerevisiae , S. paradoxus , S. kudriavzevii , C. glabrata , K. lactis , Y. lipolytica and S. pombe with H. sapiens ZNF598. The two crosslinked amino acids and corresponding amino acids in homologues are highlighted in yellow. Conserved amino acids are shown in blue (conservation w.r.t. S. cerevisiae )

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Sequencing

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2. RING and LIM domains and crosslinked amino acids are highlighted. Representation approximately in scale. b Read distributions compared between four replicates of CRAC with (wild-type) Hel2-HTP and untagged controls, showing total reads per transcript class in each data set, with fold-enrichment (of total RNA reads) in Hel2-HTP CRAC compared to untagged control indicated above corresponding bars. See also Supplementary Table

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Construct, Control

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Hel2 ribosomal crosslinking sites. a Hel2 and untagged crosslinking and analysis of cDNA (CRAC) data aligned to RDN37-1 showing reads (first and third panel) and deletions (second and fourth panel) all normalized to total reads/deletions in RDN37-1 (fifth panel). ETS external transcribed spacer, ITS internal transcribed spacer. b 18S rRNA secondary structure according to Comparative RNA Website (CRW) with stem-loop structures enriched in Hel2 CRAC highlighted in different shades of pink and crosslinked nucleotides (high-frequency micro-deletions in Hel2 CRAC) highlighted in yellow. c Crosslink sites (high frequency deletions in Hel2 CRAC) highlighted on a 3D structure representation of the S. cerevisiae ribosome. Top panel: schematic model showing the locations of the nascent peptide position and crosslinks (major: X , minor: x). Lower panel: crystal structure ; pdb 4v88, 40S subunit of monosome chain B is shown) with crosslinked nucleotides highlighted in yellow and further indicated by yellow arrows. In both panels, specific ribosomal proteins are indicated: Asc1 (dark blue), Rps3 (orange), Rps10 (red), Rps20 (violet). Other ribosomal proteins are shown in lighter grey, with rRNA in darker grey. Ubiquitination target sites are indicated by coloured arrows; violet for Rps20 (K6) (adjacent site shown, since the actual amino acid is not part of the crystal structure) and orange for Rps3 (K212)

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Effects of Hel2 mutations on binding of ribosomal RNA. a Relative distribution of reads over RDN37-1 gene encoding three of the four ribosomal RNAs. Major and minor 18S peaks and the 5.8S peak are highlighted with grey bars. The 25S peak has been seen in many different crosslinking and analysis of cDNA (CRAC) analyses and appears to be non-specific. Borders of 18S and 25S rRNAs are indicated as dashed lines. The sum of all reads over RDN37-1 is normalized to 1 for each sample. See Supplementary Fig. for schematic depiction of mutants. b Pearson correlation between binding patterns (reads) in different samples. c Change in relative fraction of deletions at previously identified crosslink peaks in 18S rRNA. Minor (U494, U1362) and major (C1490-A1492) peaks are represented. The sum of all deletions over RDN37-1 was set to 1 for each sample. Medians are only shown for samples of p < 0.05 (two-tailed Student’s t test, w.r.t. wild-type). See also Supplementary Fig. . For U494, a reduced read density was visible in a , but the frequency of deletions was not significantly altered, owing to experimental variability in deletion density for the wild-type (Supplementary Fig. )

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Effect of ASC1 deletion on Hel2 18S binding and comparison to truncation mutants. As Fig.

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Binding Assay, Comparison

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Assessment of ribosome and polysome association of different Hel2 mutants. a Representative A 260 profiles (each curve normalized to a sum of 1) of sucrose density gradients from wild-type and mutant strains (see b for colour code and Supplementary Fig. for triplicate quantification of signal in different fractions. b Immunoblot analysis of tagged Hel2 content in pooled and precipitated sucrose fractions containing different sub-populations of ribosomal components, monosomes or polysomes. Membranes were probed for the HTP-tag (using peroxidase-anti-peroxidase) and for Pgk1 as a control. c Quantification of tagged Hel2 content in each pooled fraction. Data were obtained from three independent biological replicates per strain (independently isolated clones). * p < 0.05, according to Student’s t test (two-tailed, paired, w.r.t. wild-type). See b for colour code

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Mutagenesis, Western Blot, Control, Isolation, Clone Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Assessing functional consequences of HEL2 deletion and mutation using a reporter system for ribosome-associated quality control (RQC) and no-go decay (NGD). A published reporter system encoding green fluorescent protein (GFP), TEV protease site, followed by a stalling (R12) or non-stalling ((ST)6) sequence and by red fluorescent protein to assess RQC and NGD. Scheme adapted from ref. . b Western blot for one of the three biological replicates from independent clones is shown. Data are quantified in Supplementary Fig. . Upper panel: probed with anti-GFP to visualize the reporter. Lower panel: anti-Pgk1 loading control. Migration positions are indicated for the stalled peptide (stalled) and for this peptide modified by addition of a CAT-tail (CAT). Also indicated are the major product from read-through of the stalling sequence (read-through) and the full-length protein (FL). The region around the FL product is shown in higher contrast in Supplementary Fig. and the region containing stalled and CAT products in Supplementary Fig. . Northern blot analysis of stalling and non-stalling reporter transcripts in ski2∆ background using probe Np_GFP and loading control using probe Np_scR1. Autoradiograph for one of the three biological replicates from independent clones is shown. Data are quantified in Supplementary Fig. . Migration positions are indicated for the full-length mRNA (FL), the expected 5’ product of mRNA cleavage at the stall (5’ fragment/5’-frag.) and for heterogeneous, 5’ truncated RNAs (truncated)

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Functional Assay, Mutagenesis, Control, Sequencing, Western Blot, Clone Assay, Migration, Modification, Northern Blot, Autoradiography

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: mRNA binding by Hel2. a Abundance of protein-coding genes in Hel2 crosslinking and analysis of cDNA (CRAC) data sets (median of 6 data sets for each gene) compared to RiboSeq (1 data set; from ref. ) and RNASeq (median of 3 data sets; from ref. ). The latter two were from isogenic wild-type strains. All axes are in log2. Spearman correlation between abundances of protein-coding genes in RNASeq, Hel2 CRAC and RiboSeq (medians) is shown in the right panels. b Genes were grouped into quintiles according to Hel2 CRAC read counts, in hits per million per kilobase. These are plotted against the ratio of RiboSeq reads and RNASeq reads for each gene in the quintiles. Centre lines of box plots show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, data points are plotted for outliers. c – e Metagene analysis of collapsed data sets performed for the subset of top 275 reproducibly Hel2-bound protein-coding genes with mRNA length of ≥500 nt. c , d Total reads mapped across open reading frames (ORFs) aligned by the translation start or stop codons. e Micro-deletions mapped across stop codons (smoothed data shown here, unsmoothed data shown in Supplementary Fig. ). f , g RiboSeq footprints mapped across ORFs aligned by the translation start or stop codons. The values for “% reads” or “% deletions” shown are normalized to the sum of total Hel2 and no tag CRAC signal ( c – e ) or to the sum of all RiboSeq footprints ( f , g ) over the top reproducibly bound genes

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

doi: 10.1038/s41467-019-08382-z

Figure Lengend Snippet: Effect of Hel2 mutations and asc1 ∆ on Hel2 mRNA crosslinking. Effects determined for top 252 reproducibly Hel2-bound mRNAs of transcript length >500 nt and open reading frame length >350 nt. a Metagene representation of read density over mRNAs from Start −100 nt to Stop +250 nt with nucleotides between start codon +250 nt and stop codon −100 nt (inner dashed lines) scaled. Data were separated into bins of 10 nt for non-scaled data and an additional 50 bins for scaled regions. For each data set, and for each gene, data were normalized to total read density over the gene. After summing up normalized data for each data set, read counts were then scaled according to the ratio of total read density of the respective data set and corresponding wild-type data set. The sum under the curve was set to 1 for wild-type and the other curves were scaled accordingly. Plots represent medians of normalized, scaled data from all available data sets. mRNAs were scaled between start codon +250 nt and stop codon −100 nt. Horizontal lines indicate where data were summed to give values for quantitation shown in b . b Sums of read densities after start codon (60 nt/6bins after Start codon), before stop codon (−90 to −30 nt/6 bins) and after stop codon (+50 to +110 nt/6 bins). Bars represent medians; circles represent data points from separate biological replicates

Article Snippet: Fig. 2 Hel2 construct for crosslinking and analysis of cDNA (CRAC) and read distribution. a C-terminally His 6 -TEV-ProteinA 2 (HTP)-tagged Hel2.

Techniques: Quantitation Assay