Fig 2: Rapid Depletion of EXOSC10 or DIS3 via the Auxin-Inducible Degron(A) Schematic showing the CRISPR strategy for modifying gene loci. Two repair cassettes were generated containing the AID tag, a P2A cleavage site, and either the hygromycin or neomycin resistance marker, followed by an SV40 PAS. These were flanked by 5′ and 3′ homology arms for the gene of interest.(B) Western blotting of EXOSC10 in either parental Tir1-expressing HCT116 (HCT116:TIR1) or EXOSC10-AID cells. A time course of auxin addition was applied to the EXOSC10-AID cells. Equal loading is shown by the presence of a non-specific product (∗) on the same blot.(C) Western blotting of DIS3 in either HCT116:TIR1 or DIS3-AID cells treated or not treated for 60 min with auxin. Tubulin was probed for as a loading control. Quantitative reverse transcription and PCR -derived levels of DIS3 mRNA also shown (including standard deviations [SDs]), obtained following normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels.(D) Western blotting of DIS3 in either HCT116:TIR1 or DIS3-AID cells treated or not treated for 60 min with auxin using an antibody to the AID tag. Tubulin was probed for as a loading control.(E) Co-immunoprecipitation (coIP) of GFP-MTR4 and EXOSC2 in HCT116:TIR1 or DIS3-AID cells. Input (5%) and IP are shown. Blots were probed with α-GFP (to detect GFP-MTR4) or α-EXOSC2.(F) Western blotting of EXOSC10, DIS3, MTR4, EXOSC2, EXOSC3, and as a loading control, CPSF73 in HCT116:TRI1, DIS3-AID, or EXOSC10-AID cells treated or not treated with auxin (1 h). Due to the similar size of some of these proteins, multiple blots were probed rather than using stripping. Equal loading was confirmed by loading control or ponceau. Pictures of individual blots are deposited at Mendeley (see Method Details).

Fig 3: Analysis of Redundancy between EXOSC10 and DIS3(A) Quantitative reverse transcription and PCR analysis of PPM1G, SEPHS1, and RBM39 PROMPTs in DIS3-AID or EXOSC10-AID cells treated or not treated with auxin for 4 and 8 h. Levels are expressed as fold change compared to untreated cells (dotted line) following normalization to GAPDH mRNA. n = 3. ∗p < 0.05 for differences concluded on in the text. Error bars show SDs.(B) coIP of EXOSC10 or EXOSC2 with GFP-MTR4 in EXOSC10-AID cells treated or not treated with auxin (2 h). Input and IP are shown with blots probed with α-GFP (to detect GFP-MTR4), α-EXOSC10, or α-EXOSC2.(C) Quantitative reverse transcription and PCR analysis of PPM1G, SEPHS1, and RBM39 PROMPTs in EXOSC10-AID cells treated or not treated with auxin for 24, 48, or 72 h. Levels are expressed as fold change compared to untreated cells (indicated by dotted line) following normalization to GAPDH mRNA. n = 3. ∗p < 0.05. Error bars show SDs.(D) Quantitative reverse transcription and PCR analysis of PPM1G, SEPHS1, and RBM39 PROMPTs in HCT116:TIR1 cells treated or not treated with auxin for 72 h. Levels are expressed as fold change compared to untreated cells (indicated by dotted line) following normalization to GAPDH mRNA. n = 3. Error bars show SDs.(E) Quantitative reverse transcription and PCR analysis of PPM1G, SEPHS1, and RBM39 PROMPTs in DIS3-AID cells transfected with control or EXOSC10-specific siRNAs before treatment or no treatment with auxin (1 h). Levels are expressed as fold change compared to control siRNA transfected cells not treated with auxin following normalization to GAPDH mRNA. n = 4. ∗p < 0.05 for differences concluded on in the text. Error bars show SDs.(F) EXOSC10 immunofluorescence in untreated DIS3-AID cells or the same cells treated with auxin for 1, 2, 3, or 4 h. The same cells stained with nucleolin are also shown. The red arrowheads show EXOSC10 puncta that do not overlap with nucleolin signal.

Fig 4: Rev determines the MATR3 and MTR4 binding to viral RNA.Primary CD4+ T cells were isolated from blood of healthy donors and infected with WT pNL4.3 HIV particles or Rev-mutated pNL4.3 HIV particles and collected after 3 days and used for (a) DNA extraction to quantify total HIV DNA by qPCR, for (b) total RNA extraction used for RT-qPCR quantification of RRE-containing RNA and Tat HIV-1 RNA, for (c) nuclear-cytoplasmic fractionation protocol followed by quantitative real-time RT-PCR for Tat and (d) RRE HIV-1 RNA, for (e) RNA immunoprecipitation using anti-MATR3, anti-MTR4 and anti-IgG isotype antibodies. RNA was purified from IPs and subjected to RT-qPCR targeting RRE-containing HIV-1 RNA. Values were normalized to input and shown as fold enrichment of IgG control. f–h Jurkat T cells were infected with WT pNL4.3 HIV particles or Rev-mutated pNL4.3 HIV particles and collected after 3 days and used for (f) DNA extraction to quantify total HIV DNA by qPCR, for (g) total RNA extraction used for RT-qPCR quantification of RRE-containing and Tat HIV-1 RNA and (h) RNA immunoprecipitation using anti-MTR4 and anti-IgG isotype antibodies. RNA was purified from IPs and subjected to RT-qPCR targeting RRE-containing HIV-1 RNA. All results are shown as mean values ± SEM from n = 5 (a), n = 7 (b), n = 4 (c, d, f, g), n = 6 (e), n = 3 (h) biological replicates in duplicates. Statistics were performed using a two-sided Wilcoxon signed-rank test for HIV integration (a), a two-way ANOVA test (b–g), and a two-tailed unpaired Student’s t test (h). Significant p values are indicated by the asterisks above the graphs p ≤ 0.05 [*], p ≤ 0.01 [**], ≤0.001 [***], ≤0.0001 [****]. Source data are provided as a Source Data file.

Fig 5: MATR3/MTR4/Rev ribonucleoprotein complex regulates the fate of Rev-dependent HIV-1 RNA.a 293 T cells were transfected with MS2-tagged pHIV-INTRO, pTat-GFP, and pMS2-FLAG and after 24 h whole cell lysates were subjected to anti-FLAG immunoprecipitation. b, c 293 T cells were transfected with pFLAG-MATR3, pHIV-INTRO, pTat, and pRev-GFP plasmids and after 24 h whole cell lysates were subjected to anti-FLAG (b), anti-MTR4/anti-IgG (c) immunoprecipitation followed by ±benzonase treatment or (d) anti-FLAG immunoprecipitation followed by elution with FLAG peptides for subsequent IP using either anti-IgG or anti-GFP antibodies. GAPDH is a loading control in input samples and indicates the specificity of IP. ECFP expressed from pHIV-INTRO is a transfection efficiency control in input samples and indicates the specificity of IPs. IgH (in b) refers to the immunoglobulin heavy chain from anti-flag mouse antibodies that are attached to beads. This was detected with a secondary anti-mouse antibody during the anti-GFP immunoblotting. e J-Lat 9.2 cells were stimulated with TNFα [10 ng/ml] and subjected to RNA FISH and immunostaining using antibodies against MTR4 and MATR3 for subsequent confocal microscopy analysis. MATR3 is shown in green, MTR4 in red, gagHIV-1 RNA in purple, and DAPI-stained nucleus in blue. Bright, large gagHIV-1 RNA spot corresponds to viral transcription site as described previously28,29,42. White arrows indicate triple colocalization sites. Scale bar = 10 μm. f Colocalization between MATR3-HIV-1 RNA, MTR4-HIV-1 RNA, MATR3-MTR4, and MATR3-MTR4-HIV-1 RNA was quantified by counting the number of colocalizing spots and their volumes from z-stacks obtained from 10 images/biological repetition, n = 3. Results are presented as box and whiskers with 5–95% confidence interval. Median value is shown as a bar, dots are points outside whiskers representing outliers, mean value is shown as “+”. Statistics were performed using a two-tailed unpaired Student’s t test. Statistical comparisons are indicated if p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****), “ns” indicate no significance. g–i MTR4 depletion in 293 T cells was obtained using siRNA transfection. After 24 h, cells were transfected with plasmids pHIV-INTRO, pTat-GFP, and pRev-GFP and after 24 h collected for (g) total RNA isolation, (h) nuclear-cytoplasmic fractionation protocol followed by RT-qPCR for RRE-containing, TAR, and MS HIV-1 RNAs normalized using gapdh primers and presented as relative fold changes to the values measured in non-targeting control (siCTRL) condition, which was arbitrarily set at a value of 1. i total RNA isolation followed by RNA immunoprecipitation (IP) protocol using anti-MATR3 and anti-IgG isotype antibodies. RNA was purified from IPs and subjected to RT-qPCR targeting RRE-containing HIV-1 RNA. Values were normalized to input and shown as fold enrichment over IgG control. All results are shown as mean values ± SEM from n = 5 (g), n = 3 (h), n = 3 (I) biological replicates in duplicates. Statistics were performed using a two-tailed paired Student’s t test, p ≤ 0.5 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). Source data are provided as a Source Data file.