Fig 1: TASOR recruits RNA-degradation factors onto elongating RNAPII to silence gene expression.a TASOR colocalizes with active HIV-1 transcriptional centers in HIV-1-infected Jurkat cells. Latently HIV-1-EGFP-infected Jurkat cells were transduced with VLPs containing Vpx R42A or WT for 24 h and treated with TNF (1 ng/mL). LTR-driven unspliced (us) RNAs were marked with egfp probes and TASOR by immunofluorescence. The graph corresponds to the signal intensities across the white line. b Vpx WT triggers a significant loss of TASOR at the HIV-1 transcriptional centers. TASOR signal at HIV-1 transcription centers in the Vpx R42A delivery condition was set as 100%. (n = 261, n = 267, and n = 66 for the Vpx R42A, Vpx WT, and secondary antibody (2nd.ab) Alexa 594 conditions, respectively; two-sided unpaired t test was applied: ****p < 0.001). c TASOR interacts with endogenous RNAPII in HeLa HIV-1 LTR-?TAR-Luc cells. GAPDH is a negative control (n > 3). d TASOR affinity for RNAPII is greatly reduced upon depletion of the N-terminal PARP domain. Lamin B1 and GAPDH are negative controls (n = 3). e TASOR interacts with elongating RNAPII in HEK293T and HeLa HIV-1 LTR-?TAR-Luc cells. Ser5-phosphorylated and Ser2-phosphorylated RNAPII are markers of RNAPII in the initiating and elongating phases of transcription, respectively, MPP8 is a positive control. GAPDH is a negative control (n > 3). f TASOR recruits CNOT1 and its partners onto RNAPII. DDK and TASOR-DDK vectors were transfected in HeLa HIV-1 LTR-?TAR-Luc cells. TASOR was revealed either with an anti-DDK (TASOR-DDK) or with an anti-TASOR to detect the endogenous protein. SUPT6H is a control of a known RNAPII partner. MORC2 is necessary for HUSH-mediated gene silencing according to ref. 12. GAPDH is a negative control (n = 3). g TASOR and CNOT1 are found in complex with the HIV-1 nascent transcript. Nascent RNAs from HeLa HIV-1 LTR-?TAR-Luc cells were labeled and anti-TASOR and anti-CNOT1 immunoprecipitations were performed from the nuclear extract. GAPDH and TASOR partner MATR3 are markers of the cytoplasmic and nuclear fractions, respectively. Immunoprecipitated RNAs were normalized on input signal (n = 4; mean and SEM are shown; two-sided unpaired t test was applied: purple stars or NS indicate statistical differences between TASOR and CNOT1 conditions; blue stars: TASOR vs IgG, red stars: CNOT1 vs IgG). Source data are provided as a Source data file.
Fig 2: TASOR interacts and cooperates with nuclear RNA degradation factors.a TASOR is a nuclear protein. Cytoplasmic and nuclear protein extracts from HeLa HIV-1 LTR-?TAR-Luc cells were loaded on a SDS-PAGE gel. GAPDH and MATR3 are markers of the cytoplasmic and nuclear fractions respectively (n > 3). b Endogenous TASOR interacts with the endogenous, nuclear CNOT1, and its partners CNOT7, YTHDF2, the endogenous TRAMP-like/NEXT/PAXT component MTR4, and the endogenous RNA exosome factor EXOSC10. HeLa HIV-1 LTR-?TAR-Luc cells were fractionated and endogenous TASOR immunoprecipitation was performed in the nuclear fraction. Lamin B1 is negative control (n > 3). c Reverse immunoprecipitation confirmed the interaction between endogenous CNOT1 and MTR4 proteins with endogenous TASOR in the nucleus. HeLa HIV-1 LTR-?TAR-Luc cells were fractionated and endogenous CNOT1 and MTR4 immunoprecipitation was performed in the nuclear fraction. The asterisk shows the SUPT6H band (n = 3). d TASOR interacts with the known CNOT1 partners in an RNA-dependent manner. DDK and TASOR-DDK vectors were transfected in HeLa HIV-1 LTR-?TAR-Luc cells. After 48 h, lysates were treated or not with RNase A for 30 min at room temperature. Anti-DDK immunoprecipitation was then performed. All lanes are from the same gel (n > 3). e TASOR cooperates with the nuclear RNA destabilization/degradation factors. After 72 h of siRNA transfections in HeLa HIV-1 LTR-?TAR-Luc, cells were lysed and luciferase activity was measured and normalized on protein concentration (n = 5 for the siRNA TASOR and siRNA CNOT1 conditions; n = 3 for other siRNA transfections; each color represents one different independent experiment, mean and SEM are shown). f Schematic representation of RNA-mediated interactions between TASOR and CNOT1 and its partners: the TRAMP-like/NEXT/PAXT component MTR4, the Nuclear Exosome, and the m6A reader YTHDF2. Source data are provided as a Source data file.
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