EMBO/soda-nel huggingface dataset:
Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites (2020)
Figure 6

Figure 6: Effect of GS inhibitor on NF54 and NF135

A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains).

B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001.

C) Number of schizonts per well for NF135 (green) and NF54 (blue). Three biological replicates (circle, square and triangle), each with two technical replicates were performed.

D) Average +/- s.d. (dot) host cell numbers per well for three independent biological replicates under the different AIP concentration.

E) The difference in size between GS positive (closed circle) and negative (open circle) NF135 schizonts (day 5 p.i.) after treatment of AIP (360 µM) on different days pre - and post-invasion. Each dot represents the median size of ≥100 schizonts +/- s.d.. The dotted blue line shows the average median schizont size of NF54 treated with H2O on day 5 post invasion of the same three biological replicates. A two-way RM ANOVA was performed followed by a Tukey's multiple comparison test: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001).

F) Summary of observed effects of hGS on parasite development. Sporozoites can invade Z1, 2 and 3 hepatocytes but disproportionally prefer Z3 hepatocytes to make use of exclusive presence of hGS. Ability to uptake functional hGS early after invasion results in relatively larger schizonts; uptake of inhibited hGS (ihGS) reduces schizont size. (See Appendix Fig S3-5 for raw measurements per replicate).

Expanded view figure legends
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 1

Figure 1: hnRNPL binds and stabilizes reporter transcripts.

A The first 400 nt of all 3'UTRs represented in the UPF1 RIP-Seq dataset (Lee et al, 2015) were analyzed for the occurrence of motifs corresponding to the indicated RNA-binding proteins. Transcripts were divided into groups based on the number of motifs present (0, 1, more than 1), indicated by the gradient triangle. The dotted line indicates the mean RIP-Seq efficiency for all transcripts used in the analysis. Mean and SD are plotted for all groups.

B CDF plot of relative abundance of transcripts with 3'UTRs > 1000 nt in K562 cells under conditions of UPF1 or non-targeting shRNA, for transcripts with hnRNP L eCLIP peaks in the indicated intervals (Sundararaman et al. 2016). Each trace represents a category of CLIP peak locations. For mRNAs with peaks > 400 nt from the TC or with peaks in the 5'UTR or CDS, transcripts with peaks at 3'UTR positions 1-400 were excluded. Statistical significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons and was determined based on comparison to the set with no 3'UTR CLIP peaks.

C Representative northern blots assaying the stability of β-globin reporter transcripts containing the SMG5 3'UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet-off cells. Half-lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint. Significance was measured compared to the SMG5 3'UTR reporter using the sum-of-squares F test.

D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from (C). "AS" indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3'UTR in the same position as the RSE. The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation. * indicates P = 0.0146 in two-tailed Student's t-test. The reporter containing the GAP 3'UTR used here as a transfection control contains a predicted hnRNP L binding site, allowing its consistent recovery in the IPs.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 2

Figure 2: hnRNPL protects select human transcripts with long 3'UTRs from NMD.

A RNA-seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1-dependent manner.

B CDF plot of transcript 3'UTR lengths for subsets identified in (A). Statistical significance was determined by two-sided K-S test. P value for decreased set is from comparison to the set of total transcripts. P value for rescued set is from comparison to decreased set.

C Venn diagram (to scale) of populations of mRNAs putatively protected from NMD by hnRNP L or PTBP1.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 3

Figure 3: hnRNP L confers a spectrum of protection from NMD

A qPCR of selected transcripts found by RNA-Seq to decrease in abundance in the absence of hnRNP L. Values represent the mean and standard deviation of 3 independent biological replicates. Significance of hnRNP L KD and UPF1 KD were compared to NT, and the double knockdown was compared to hnRNP L KD. For clarity, only a single star is used to indicate significance; in all cases P ≤ 0.025 in two-tailed Student's t-tests.

B Half-lives of selected transcripts under conditions of non-targeting or hnRNP L knockdown. Measurements were calculated based on proportional recovery of transcript labeled with 5-EU in 1 hour versus total transcript. Significance was determined using two-tailed Student's t-test: *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Values represent mean and standard deviation of 3 independent replicates.

C The stability of β globin reporters with the CSRP1 3'UTR, along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated, were evaluated in HeLa Tet-off cells. The schematic indicates areas of 3' UTR CA-enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3'UTR, where putative hnRNP L binding elements are in red typeface. The northern blot is a representative example from 3 independent replicates. Mean values plotted with SD at each timepoint were used to quantify decay (right panel). Significance is measured compared to the WT reporter using the sum-of-squares F test.

D The stability of the WT and L mut 1-200 CSRP1 reporters in HeLa Tet-off cells treated with non-targeting RNAi or siRNA against UPF1 was evaluated. The northern blot is a representative example from at least 3 biological replicates. Decay quantification and statistics were performed as in (C), with significance measured compared to the non-targeting condition.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 4

Figure 4: hnRNP L and PTBP1 motifs enhance predictive utility of 3'UTR length.

A Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq (Lee et al, 2015). Transcripts were binned into three 3'UTR length classes, which were then further subdivided based on hnRNP L motif density. Each box plot represents the median and interquartile range with extensions from 10% to 90% of the indicated subset. Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and no hnRNP L binding motifs. Significance was computed using Kruskal-Wallis tests with Dunn's correction for multiple comparisons.

B CDF plots of selected transcript classes from (A). Log2(FC) of UPF1 knockdown as compared to the non-targeting control indicates NMD sensitivity of each class. Significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons

C Motif analysis of recovered UPF1-associated transcripts analyzed by RIP-Seq (Lee et al, 2015). Transcripts are divided into tertiles by hnRNP L and PTBP1 binding motif density and 3'UTR length. Data are represented and analyzed as in (A). Dotted line represents the median RIP-Seq efficiency of the class of transcripts with the shortest 3'UTR and low PTBP1/hnRNP L motif density.

D CDF plots of selected transcript classes from (C) analyzed as in (B).

Data information: **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 5

Figure 5: hnRNPL protects the Bcl2 fusion transcript from NMD.

A Schematic of normal and fusion BCL2 transcript splicing. Introns are indicated with chevrons. Double slashes indicate segments that have been abridged for simplicity. The light blue region of the 3'UTR of BCL2 represents its first 400 nucleotides, with the sequence provided below. Nucleotides in red indicate stretches of hnRNP L recognition sequence.

B Schematic of aberrant BCL2:IGH mRNAs generated by the t(14;18) translocation event found in many B cell lymphomas. Dotted chevrons indicate alternative events that generate either the secreted or membrane-bound form of IgH.

C qPCR of selected transcripts in SU-DHL-4 cells under conditions of non-targeting RNAi or hnRNP L knockdown with or without 50 μg/mL cycloheximide treatment (for 4 hours; n ≥ 3). Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO, and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO. Bars represent the mean ± SD. For clarity, only a single star was used to indicate significance; in all cases P ≤ 0.05 in two-tailed Student's t-tests.

D Schematic of the reporter constructed to assay protection of the fusion transcript, consistent with the color scheme in (B). Spliced sequences are indicated with chevrons (see Appendix Fig S3 for alignments). Bright pink indicates the AdML major-late intron used in place of the long intron between J and the first Cγ IGH exon. Only exons encoding secreted IgH were used.

E Half-life determination from RT-qPCR detection of reporter constructs in HeLa Tet-off cells transfected with non-targeting or UPF1 siRNAs. For plots comparing non-targeting with UPF1 knockdown, significance is compared to the non-targeting condition using the the sum-of-squares F test. The fourth plot compares the non-targeting decay curves of the three reporters, and significance is compared to the decay of the fusion transcript. Points represent the mean ± SD from qRT-PCR at each time point of at least 3 independent experiments.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 6

Figure 6: hnRNP L depletion results in increased apoptosis in SU-DHL-4 cells.

A qPCR of selected transcripts in SU-DHL-4 cells under conditions of BCL2 knockdown, hnRNP L knockdown, or non-targeting control siRNA Nucleofection (n ≥ 4 independent experiments). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests.

B Quantification of protein reduction in knockdown experiments and a representative western blot. Each point represents a trial (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot. Target protein levels from all biological replicates were normalized to MATRIN3, with statistical analysis as in panel (A).

C FACS-based apoptosis analysis of SU-DHL-4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours. Quantification of relative change in each quadrant is provided where points represent individual experiments (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel (A). A representative FACS experiment is shown with the percentage of cells in each gate indicated.

Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma (2018)
Figure 7

Figure 7: Deletion of hnRNP L binding site in the BCL2 3'UTR results in increased apoptosis in SU-DHL-6 cells.

A Schematic of deleted region in CRISPR/Cas9-edited cells. A portion of the 3'UTR at the BCL2 locus is represented, with the last exon as a wide box and the 3'UTR as a narrow box. The light blue region of the 3'UTR represents its first 400 nucleotides, in which the relevant hnRNP L binding sites are located. The "X" indicates the region of the deletion.

B SU-DHL-4 and SU-DHL-6 cells were treated with varying doses of BCL2 inhibitor. Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells. Traces were generated from at least 5 independent biological replicates. Points represent mean ± SD at each dose. P value from comparison of dose response between SU-DHL-4 and SU-DHL-6 cells calculated using sum-of-squares F test is given in the graph key. Significance of individual points calculated using two-tailed Student's t-tests is marked using asterisks on the graph.

C qPCR of selected transcripts in SU-DHL-6 cells under conditions of hnRNP L knockdown or non-targeting control (n = 5 independent experiments). Bars indicate mean ± SD. Significance of hnRNP L KD was determined compared to NT using two-tailed Student's t-tests.

D Representative amplification of the region of chromosomal deletion in edited cells. PCR products were run on a 1% agarose gel to resolve the amplicons from edited and unedited chromosomes.

E qPCR of selected transcripts in edited SU-DHL-6 cells (n = 4 independent experiments). Significance was determined as compared to control, and data are represented as in (C).

F Quantification of protein reduction and western blot of lysates from a representative experiment of edited cells. Target protein levels from all biological replicates were normalized to MATRIN3. Each point represents a trial (n = 4). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests.

G FACS-based apoptosis analysis of edited SU-DHL-6. Quantification of relative change in each quadrant is provided, where points represent individual experiments (n = 6 for a given condition). Mean with standard deviation are indicated on the plot, analyzed as in (F). Shown is a representative FACS experiment. Percentage of cells in each gate is indicated.

Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Extended View Figure Legends
Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8 (2013)
f1

(a) The ternary NDP52–GAL8–LC3C complex (green line), binary NDP52–GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS. The dotted line represents measured MW and the vertical bars (1 and 2) indicate the gap in MW. The SEC-MALS result of NDP52 alone (red line) shows that the oligomeric state is ambiguous between dimer and trimer. However, the NDP52–GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone, which confirmed that two molecules of monomeric GAL8 (MW: ~33.5 kDa; Supplementary Fig. S1b) bound to homodimeric NDP52. Furthermore, the ternary NDP52–GAL8–LC3C complex showed a MW difference of ~30 kDa compared with the binary NDP52–GAL8 complex, which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex. (b) Size-exclusion chromatography data of NDP52–GAL8–LC3C complex. The first peak (marked by an orange bar) represents the ternary complex, as shown by the SDS–polyacrylamide gel electrophoresis (SDS–PAGE) results marked by an orange bar (lane 1, 2 and 3). The second merged peak (marked by green and yellow bar) and corresponding SDS–PAGE results show the remaining free GAL8 (lane 4, 5, 6 and 7) and LC3C (lane 5, 6, 7 and 8). (c) A schematic model of an NDP52 dimer with two LC3Cs (orange) and two GAL8s (yellow). Each monomer is distinguished by red and blue colour, and the ‘PGLAYGNPY’ indicates the amino-acid sequence of the Galbi loop.
Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8 (2013)
f2

(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).
Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8 (2013)
f3

(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a–c) Red dashed lines designate hydrogen bonds. (d) An FP assay using FITC-labelled Galbi peptides, including mutants (L374A, Y380F and Y380A) against an increasing concentration of GAL8. (e) An FP assay using a FITC-labelled Galbi peptide against an increasing concentration of GAL8 mutants (E268A, I270A, Y272A and Trp317). (d,e) The KD values were calculated from at least three independent experiments. ND, not determined.
Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8 (2013)
f4

(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.
Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8 (2013)
f5

(a) Three GAL8 structures, including both CRDs, are available from PDB (4FQZ—lactose bound in only N-CRD; 3VKL—lactose bound in both N-CRD and C-CRD; 3VKM—sialylated lactose in N-CRD and lactose in C-CRD). The C-CRDs of these Gal8 structures were superposed with that of our Gal8. For clarity, only Cα-traces of each structure (our GAL8—yellow; 4FQZ—red; 3VKM—blue; and 3VKL—cyan) are shown. The large rotation of the N-CRD domain relative to C-CRD compared with the other GAL8 structures is observed in the GAL8–NDP52 complex. A representative angle between N-CRD of our GAL8 and that of the 4FQZ structure is indicated in degrees. (b) A 90° rotation of panel (a) along the horizontal axis showing a top view. Arg59, which is a unique residue of Gal8 N-CRD that provides carbohydrate specificity, is used as a standard for measuring the distance of domain movement.
Gut colonization by Bacteroides requires translation by an EF-G paralog lacking GTPase activity (2022)
Figure 2

Figure 2. B. thetaiotaomicron EF-G2 supports slower translation elongation than B. thetaiotaomicron EF-G1 and E. coli EF-G

A. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.

B. Ribosome translocation determined as the fluorescence change of the fluoresceine-labelled mRNA in stopped-flow experiment supplemented with the indicated proteins and nucleotides. Translocation rates are as follows: EF-G1 with GTP, 6.9 ± 0.1 s-1; EF-G1 with GDPNP, 0.64 9 ± 0.01 s-1; EF-G2 with GTP, 0.7 ± 0.01 s-1; EF-G2 with GDPNP, 0.62 ± 0.01 s-1. See Methods for details. Shown are averages of 5-7 technical replicates.

C. Tripeptide formation assay using translation initiation complex programed with the tripeptide-encoding mRNA, corresponding aminoacyl-tRNA:EF-Tu:GTP ternary complexes and GTP, and the indicated EF-G proteins following incubation for 1 or 5 min. Shown are the results from two independent experiments and their average. Insert: tripeptide formation with EF-G2 following incubation for 0, 1, 5, and 10 min.
Gut colonization by Bacteroides requires translation by an EF-G paralog lacking GTPase activity (2022)
Figure 4

Figure 4. Biochemical features of B. thetaiotaomicron EF-G2

A. Structure of EF-G2 domain IV highlighting the insert (residues 514-539) and hairpin (residues 516-527) regions.

B. Western blot analysis of reporter HslO-FLAG protein synthesized in vitro using a custom-made PURExpress® system with EF-G1, EF-G2, and two engineered EF-G2 variants: one missing the 26-amino acid insert, EF-G2 (∆514-539) and one missing the beta hairpin in the insert, EF-G2 (∆516-527). Shown is a representative experiment from at least two independent experiments.

C. GTP hydrolysis by EF-G1, EF-G2 and indicated variants incubated in the presence of vacant E. coli ribosomes. Shown are results from three technical replicates and their average, error bars correspond to SD, and p-values derived from two-tailed Student's t-test comparing each protein to the blank.

D. SDS-PAGE analysis of co-sedimentation assays monitoring factor (EF-G1 EF-G2 or EF-G2 variant EF-G2 (∆516-527) or EF-G2 (∆514-539)) binding to E. coli 70S ribosomes. Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets. The positions of EF-G proteins and ribosomal proteins and molecular weight markers are indicated.

E. Western blot analysis of reporter HisC-FLAG protein synthesized in vitro using a custom-made PURExpress® system with the EF-G1, EF-G2 or EF-G2(S84H) proteins. Shown is a representative from at least two independent experiments.