Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, SRA SRA analysis. a Schematic illustration of the experimental approach. Repeated AGPC extraction enriches RBPs over non-RBPs; effect of repeated AGPC extraction on S / N was tested. Graphical representation of SRA: RNase-treated samples are compared to equivalent amounts of untreated samples by SDS-PAGE. RNA-bound proteins in untreated samples migrate at a higher molecular weight than their unbound counterparts. RNase digestion liberates RNA-bound RBPs allowing their mobilization into the separating gel. b Comparison of AGPC interphase samples isolated by methanol precipitation (95% v/v) following 1–6 AGPC extraction(s) of UV-crosslinked or non-crosslinked HeLa cells by SRA and Coomassie Blue (protein), SYBR Safe (RNA&DNA) staining; parallel gels. Protein (BCA) and RNA&DNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. c Immunoblot analysis of samples from ( b ). Sample compositions, target protein RBP–RNA interactome category, and target protein GO:RBP-annotation status indicated in the figure panel. Full bots are provided in Supplementary Fig. , RNA-binding domains and list of studies reporting UV-enrichment* provided in Supplementary Data . d Schematic illustration of enhanced S / N output. SRA is unable to distinguish non-RBPs from RBPs with low S / N in RNP fractions isolated by methanol precipitation (95% v/v) from the final AGPC interphase. SRA supports the identification of RNA enrichment methods that further enhance S / N , evidenced by a marked decrease in RNase-insensitive protein. Graphics ( a , b ) were created with BioRender.com. Experiments ( b , c ) were performed four times with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Extraction, SDS Page, Molecular Weight, Comparison, Isolation, Staining, Spectrophotometry, Western Blot, RNA Binding Assay

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, M methanol precipitation (95% v/v), I INP, L LEAP-RBP, SRA SRA analysis. a Comparison of RNP fractions isolated by M, I, and L methods from final AGPC interphase suspensions of UV-crosslinked cells by SRA and Coomassie Blue (protein), SYBR Safe (RNA&DNA) staining. Protein (BCA) and RNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. Effect of isolation method on protein yield analyzed using one-way ANOVA (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): significant main effect of isolation method (m*), F (2, 6) = 41.91, P < 0.001 (MvI*, P = 0.018; IvL*, P = 0.001; MvL*, P < 0.001). Effect of isolation method on RNA yield analyzed using a one-way ANOVA (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): non-significant main effect of isolation method (n.s.), F (2, 6) = 0.04, P = 0.965 (post hoc testing not applicable). b Immunoblot analysis of samples from a , sample compositions indicated in figure panel. c Pictorial representation of LEAP-RBP method: (A) Addition of chloroform and vortexing. (B) Addition of precipitation solution (LiCl and isopropanol), inversion and incubation for 1 minute; repeated 5+ times. (C) Vortexing (D) Centrifugation and rinsing with 95% methanol (v/v). Experiments ( a , b ) were performed twice with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Comparison, Isolation, Staining, Spectrophotometry, Two Tailed Test, Western Blot, Incubation, Centrifugation

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol–chloroform, L LEAP-RBP. Sample preps: s.p.1, L; s.p.2, L w/ DNA depletion step; s.p.3, repeated AGPC extraction and L w/ DNA depletion step. a SRA analysis of L fractions isolated from AGP input suspensions containing equivalent amounts of UV-crosslinked or non-crosslinked cells by s.p.1, s.p.2, s.p.3; parallel gels; Coomassie Blue (protein), SYBR Safe (RNA&DNA). Protein (BCA) and RNA&DNA (UV-spectrophotometry) yields shown in the adjoining bar charts represent the mean ± 1 SD of three biologically independent samples, pooled (equivalent % fraction) for SRA analysis. Effect of UV cross-linking and sample prep on protein yield of L analyzed using two-way ANOVA: significant interaction, F (2, 12) = 4.61, P = 0.033. Subdivided by largest main effect (m.e.), UV cross-linking (m.e.1*, dashed line), F (1, 12) = 93.66, P < 0.001. Effect of sample prep on protein yield of L for UV-crosslinked and non-crosslinked subgroups analyzed using independent one-way ANOVAs (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): non-crosslinked, significant m.e. of sample prep (m.e.2*), F (2, 6) = 6.07, P = 0.036 (e.1*, P = 0.023; e.2*, P = 0.023; n.s.1, non-significant, P = 0.998); UV-crosslinked, significant m.e. of sample prep (m.e.3*), F (2, 6) = 6.86, P = 0.028 (e.3*, P = 0.010; n.s.2, non-significant, P = 0.076; n.s.3, non-significant, P = 0.173). Effect of UV cross-linking and sample prep on RNA&DNA yield of L analyzed using two-way ANOVA: significant interaction, F (2, 12) = 719.45, P < 0.001. Subdivided by largest m.e., sample prep (m.e.4*, dashed lines), F (1, 12) = 998.29, P < 0.001. Effect of UV cross-linking on RNA&DNA yield of L for s.p.1, s.p.2, s.p.3 subgroups analyzed using independent one-way ANOVAs (Fisher’s PLSD post hoc test, unpaired, two-tailed, homoscedastic, no correction): s.p.1, non-significant m.e. of UV cross-linking (n.s.4), F (1, 4) = 0.14, P = 0.729; s.p.2, non-significant m.e. of UV cross-linking (n.s.5), F (1, 4) = 0.91, P = 0.393; s.p.3, significant m.e. of UV cross-linking (m.e.5*), F (1, 4) = 746.88, P < 0.001. b Immunoblot analysis of samples from ( a ), sample compositions indicated in figure panel. Experiments ( a , b ) were performed four times with similar results. Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Extraction, Isolation, Spectrophotometry, Sample Prep, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: AGPC acidic guanidinium thiocyanate–phenol-chloroform, I INP, L LEAP-RBP, SILAC SILAC LC–MS/MS analysis, SRA SRA analysis, E* significantly UV-enriched*, NE not E*. a Schematic illustration of the experimental approach. Heavy SILAC-labeled UV-crosslinked and light SILAC-labeled non-crosslinked cells were mixed prior to repeated AGPC extraction and isolation of RNP fractions by L or I methods. Graphic prepared in BioRender. b Volcano plots showing proteins identified as E* (red) or NE (blue) in I or L fractions by SILAC. Log 2 (CL/nCL) ratios were generated with SPI CL values and average SPI nCL values. Tested against the null hypothesis that protein log 2 (CL/nCL) ratios ( n = 3 biologically independent samples) are equal to 0 using unpaired, upper-tailed, heteroscedastic t tests (RNA-binding proteins are expected to be recovered from UV-crosslinked cells in greater amounts). Correction for multiple hypothesis testing was performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%. c GO-enrichment analysis of proteins identified as E* in I or L fractions by SILAC (Fisher’s Exact, two-tailed, correction for multiple hypothesis testing performed using the Benjamini–Hochberg approach and a false-discovery rate of 5%). d Venn diagram showing overlap of total and E* proteins identified in I and/or L fractions by SILAC. Pie charts showing the number of RBPs and non-RBPs identified as E* or NE in I or L fractions by SILAC. For information on protein ID assignment and protein lists for each category or GO term used in this study, see Supplementary Data and . e Stacked bar charts showing the number of proteins with annotated RNA-binding or RNA-related functions identified as E* or NE in I or L fractions by SILAC. f Histograms showing average log 2 (CL/nCL) ratios of RBPs and non-RBPs identified in I or L fractions by SILAC. Font color for individual proteins reflects conclusions from SRA and immunoblot analysis of total clRNP fractions; red text: RNase-sensitive RBP; blue text: undetected or RNase-insensitive protein. Asterisks: E*. I and L SILAC LC–MS/MS experiments ( a ) were performed once with three biologically independent samples for each SILAC label group (CL, nCL). Source data are provided as Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Extraction, Isolation, Generated, RNA Binding Assay, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Signal-noise metrics for RNA binding protein identification reveal broad spectrum protein-RNA interaction frequencies and dynamics

doi: 10.1038/s41467-023-41284-9

Figure Lengend Snippet: N/AGPC neutral/acidic guanidinium thiocyanate–phenol-chloroform, AGP acidic guanidinium thiocyanate–phenol, L LEAP-RBP, X XRNAX, O OOPs, P Ptex, T TRAPP, R RIC, SILAC, SILAC LC–MS/MS analysis, SRA SRA analysis, E* significantly UV-enriched*, NE not E*. a Experimental flow outlining the main steps of LEAP-RBP and five referenced RNA-centric methods. TBE gel analysis was performed on 1 µg of RNA isolated by NGPC extraction of proteinase K-treated RNP fractions isolated from UV-crosslinked cells and an equivalent % fraction of their corresponding non-crosslinked samples. UV-dependence of protein and RNA recovery as well as S / N were evaluated by SRA with SYBR Safe (RNA&DNA), Coomassie Blue (protein), and silver stain (RNA, DNA, and protein) staining ( b ) or immunoblot ( c ). Sample compositions and/or normalization values are indicated in figure panels b, c . Immunoblot targets found UV-enriched* in referenced studies (Supplementary Data ) were marked with gold asterisks in c ; black asterisk (T): no yeast homolog, hence n/a; n.d.: not detected. d Specificity and selectivity of the different methods for RNA-bound RBPs was evaluated by comparing observed abundances of RBPs and non-RBPs as a function of their log 2 ( S / N ) ratios (SILAC and non-SILAC). Differences in corresponding frequency curves plotted as a function of log 2 ( S / N ) (solid) or log 10 (%TP) (dashed) indicate differences in protein–RNA adduct enrichment efficiency ( S / N ) or abundance (%TP), respectively. e Stacked bar charts showing the number of RBPs and non-RBPs identified as E* or NE by each method, or the estimated %TP S and %TP N contributions of RBPs and non-RBPs in each RNP fraction. Experiments were performed once ( a – c ); n = 1 (X, O, P, T, R); n = 3 biologically independent samples (L), pooled (equivalent % fraction) for SRA. Input samples isolated from AGP input suspensions containing UV-crosslinked and/or non-crosslinked HeLa cells were used as inter-run controls during SRA analysis, n = 1. Complete MS datasets and parameters discussed in this study for each of the referenced studies are provided as individual Excel files (Supplementary Data 10–20). Source data are provided as a Source Data file.

Article Snippet: Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Extraction, Silver Staining, Staining, Western Blot