nat10 ko  (Proteintech)

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: ( A ) ac4C-rich RNAs localize in G3BP- and PABP-containing stress granules (SG) in response to oxidative and osmotic stresses, caused by sodium arsenite and d-sorbitol treatments, respectively. Individual SG are indicated by arrows. For panels ( A , D , E ) ac4C is depicted in green, PABP, G3BP or NCL in red, DAPI staining (nuclei) is in blue, scale bar 10 µm; ( B ) RNA mass spectrometry (RNA MS) analysis shows strong decrease in ac4C level in NAT10 KO HeLa cells. n = 3 biological replicates, data represented as Mean ± SD. Unpaired two-tailed t -test, *** p < 0.001; ( C ) Reduction in NAT10 protein level is confirmed by western blot; ( D ) Decreased ac4C IF signal in NAT10 KO cells suggests decreased ac4C levels in nucleoli of untreated and SG of arsenite-stressed cells. Individual organelles are indicated by arrows; ( E ) ac4C IF signal disappears from SG of arsenite-stressed cells in response to RNase A treatment. The levels of RNA modifications m6a, ac4C and m7G in ( F ) SG, ( G ) poly(A) RNA, ( H ) total RNA, ( I ) 18S rRNA and ( J ) tRNA from WT HeLa cells measured by RNA MS. Experiments are done in 2 replicates for panel ( G ) and three replicates for panels ( F , H – J ), data represented as Mean ± SD.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Staining, Mass Spectrometry, Two Tailed Test, Western Blot

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: Untreated WT HeLa cells stained for ac4C (green) and the nucleolus marker NCL (Nucleolin; red) ( A ) and arsenite stressed WT HeLa cells stained for ac4C and the SG marker G3BP (red) ( B ). DAPI staining (nuclei) is in blue. Arrows indicate ac4C granules overlapping with nucleoli in ( A ) and SG in ( B ). Scale bar 10 µm. In ( C ) the intensities of SG, defined from G3BP fluorescence signal of arsenite-stressed WT and NAT10 KO HeLa cells are quantified and shown as corrected total SG fluorescence. At least 25 cells are analyzed per each condition. Median (solid) and quartiles (dashed), unpaired two-tailed Student’s t -test. In ( D ) is shown the ratio between SG levels of m6A, ac4C and m7G compared to their respective levels on mRNA, measured by RNA mass spectrometry (MS). .

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Staining, Marker, Fluorescence, Two Tailed Test, Mass Spectrometry

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: Panel ( A , B ) shows volcano plots of SG RNA compared to total RNA for WT ( A ) and NAT10 KO HeLa cells ( B ) from RNA sequencing experiments done in four biological replicates. Benjamini-Hochberg adjusted p -value. We compared enriched ( C ) and depleted ( D ) transcripts with previous studies purifying SG from U2OS cells (Matheny et al, ; Khong et al, ). We assessed translation efficiency ( E ) and mRNA length ( F ) for high-confidence SG transcripts enriched >2-fold in all studies and those enriched only in HeLa cells in the present study, respectively ( n = 559 high-confidence transcripts, n = 915 transcripts unique in HeLa). Unpaired two-tailed Student’s t -test, **** p < 0.0001.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: RNA Sequencing, Two Tailed Test

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: The overlap between acetylated transcripts in WT HeLa cells and transcripts enriched in SG (53.3 per cent) is shown as a Venn diagram in panel ( A ). The transcript with the most ac4C sites, MKI67, is shown as a schematic in panel ( B ). Panel ( C ) The transcripts with more than one ac4C show tendency towards localization in SG upon arsenite-induced stress in HeLa WT cells. ac4C-transcripts are shown along with fold change enrichment in SG and normalized average expression as FPKM. Panels ( D ) and ( E ) show the cumulative distribution of the log 2 (Fold change) for mRNAs in SG compared with total mRNA. ac4C-containing transcripts are enriched in arsenite-induced SG in WT HeLa cells ( D ), and the effect increases with the number of ac4C sites. With the ac4C depletion (HeLa NAT10 KO cells) ( E ), normally ac4C-enriched transcripts are considerably less abundant in SG compared to WT. The number of mapped ac4C sites for each bin is indicated.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Expressing

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: ( A ) mRNA enrichment in SG, compared to total mRNA, is related to transcript length in arsenite-stressed WT HeLa cells. Cumulative distribution of the log 2 (Fold change) of transcripts, binned on the basis of length is shown. Compared to WT, transcript length shows considerably weaker influence on mRNA localization to SG in arsenite-stressed ac4C-depleted (NAT10 KO) HeLa cells ( B ). For the further analysis we used a cut-off of four-fold enriched in SG, and comparison of TE for this set is shown in panel ( C ) where SG transcripts are defined as having a log 2 FC <−2, and transcripts not enriched in SG are defined as having a log 2 FC >−1 ( n = 449 SG and n = 1346 non-SG), one-way ANOVA. In ( D ) are shown 8 model transcripts used for comprehensive SG studies (Khong et al, ) and their change in SG localization from WT to NAT10 cells. The arrow indicates the direction of the change and below each data point is shown the number of acetylation sites for each transcript, respectively.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Comparison

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: We used smFISH, coupled to G3BP-IF, to further substantiate our findings using the control transcript GAPDH ( A ) and core SG transcripts AHNAK ( B ) and MKI67 ( C ). Transcript localization upon arsenite stress for either 0 min, 30 min or 1 h in WT and NAT10 KO HeLa cells is shown. Red is smFISH probe, green is G3BP and blue is DAPI. Scale bar, 10 µm. Quantification of the fraction of AHNAK mRNA ( D ) and MKI67 ( E ) in SG per cytoplasm in different conditions. 20 cells per each condition were counted, each circle represents a single cell. Data is represented as Mean ± SEM, unpaired two-tailed Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. .

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Control, Two Tailed Test

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: ( A ) smFISH-IF microscopy images of poly(A) RNA and G3BP accumulation into SG of WT HeLa cells in response to treatment with 0.5 mM NaAsO 2 for 0 min, 30 min or 60 min. Arrows indicate individual SG. Poly(A) RNA is depicted in red (as well as GAPDH mRNA in ( B ), AHNAK mRNA in ( C ) and MKI67 mRNA in ( D )). For panels ( A – D ) G3BP in green, DAPI staining (nuclei) is in blue, scale bar 10 µm; smFISH-IF microscopy images of ( B ) GAPDH, ( C ) AHNAK and ( D ) MKI67 mRNA localization in response to treatment with sodium arsenite with 10 µg/ml puromycin for 0 min, 30 min or 60 min in HeLa WT and NAT10 KO cells; Quantification of the fraction of AHNAK mRNA ( E ) and MKI67 ( F ) in SG per cytoplasm in different conditions. 20 cells per each condition were counted. Data represented as Mean ± SEM. Unpaired two-tailed Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Microscopy, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: The experimental strategy to identify ac4C-binding proteins is shown in panel ( A ). Panel ( B ) shows the sequences of three RNA oligos used in the immunoprecipitation experiments. Putative NAT10 recognition motifs (CCG) are highlighted in red. Panels ( C and D ) show the RNA MS analysis of oligos, where input ac4C/C ratio was 50% during IVT (in vitro transcription) and reflect on actual amount of ac4C in these oligos. ( E ) and ( F ) show peptides used to identify NOP58 in mass spectrometry experiments.

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Binding Assay, Immunoprecipitation, In Vitro, Mass Spectrometry

Journal: EMBO Reports

Article Title: N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules

doi: 10.1038/s44319-024-00098-6

Figure Lengend Snippet: Panel ( A ) shows the change in SG enrichment from WT to NAT10 KO HeLa cells for the SG model transcripts as a function of interaction propensity with NOP58 (identified as ac4C-binder by LC/MS) predicted by Catrapid. The size of the datapoints show the number of acetylation sites on each transcript. ( B ) Dose-dependent interaction of NOP58 to acetylated RNA oligonucleotides validated by western blot (upper panel) and quantified as compared to 100% input (lower panel). GAPDH is used as a control. ( C ) RIP-qPCR of NOP58 and IgG (as negative control) interactions with the SG core transcript AHNAK as well as MKI67 and GAPDH in WT HeLa cells. Data of three biological replicates is represented as Mean ± SD, unpaired two-tailed Student’s t -test, * p < 0.05. Immunostaining of NOP58 (green) and G3BP (red) for unstressed cells are shown in panel ( D ) and for arsenite stressed cells shown in panel ( E ). Arrows indicate NOP58 granules overlapping with nucleoli in ( D ) and SG in ( E ). NOP58 containing SG are not indicated in NAT10 KO HeLa cells in ( E ) as they are not visible. DAPI staining (nuclei) is in blue. Scale bar 10 µm. Panels ( F – H ) shows quantification of NOP58 Mean Fluorescence Intensity (MFI) in nucleoli of unstressed ( F ) and arsenite stressed cells ( G ). In ( H ) is shown quantification of MFI of SG in arsenite stressed WT and NAT10 KO HeLa cells. At least 50 cells per each condition were analyzed. Median (solid) and quartiles (dashed), unpaired two-tailed Student’s t -test, **** p < 0.0001. .

Article Snippet: To confirm NAT10 KO in CRISPR/Cas9 generated HeLa NAT10 KO cell-line, anti-NAT10 (#13365-1-AP, Proteintech) antibody was used.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Negative Control, Two Tailed Test, Immunostaining, Staining, Fluorescence