Fig 2: Endogenous transcripts are subject to PABPN1-mediated hyperadenylation.(A) Schematic diagram of the in vivo labeling procedure. See text for details. (B) Bulk poly(A) tail analysis of labeled RNAs from cells transfected with indicated siRNAs. Prior to harvesting the RNA, cells were incubated with EU for 2 hr as indicated. (C) In vivo labeling experiment with cells transfected with the indicated siRNAs. Double asterisk marks extensive hyperadenylation observed upon RRP6 and DIS3 co-depletion. (D) Quantification of the poly(A) tails greater than 400 nt in length from (C). We “boxed” signals corresponding to poly(A) tails >400 nt, subtracted background signal (no EU) and normalized to the signal from the siRNA control samples.

Fig 3: 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 a-GFP (to detect GFP-MTR4) or a-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 4: Direct Detection of EXOSC10 Substrates by iCLIP(A) iCLIP trace of 5.8S rRNA locus obtained from EXOSC10WT and EXOSC10CAT samples. There is a clear enrichment of reads for the EXOSC10CAT sample showing a 30-nt “footprint” immediately beyond the 5.8S gene (indicated by vertical lines). y axis units are reads per million mapped.(B) iCLIP traces of SNORA69 and SNORD18C genes obtained from EXOSC10WT and EXOSC10CAT samples. There is strong enrichment of reads for the EXOSC10CAT sample showing a 30 nucleotide footprint immediately beyond each gene. y axis units are reads per million mapped.(C) Metagene plots of iCLIP reads over the 5' or 3' regions of snoRNA genes in EXOSC10WT and EXOSC10CAT samples. There is a clear 30-nt footprint immediately 3' of snoRNA genes.(D) IGV browser tracks of SNORA48 and SNORA68 genes in EXOSC10-AID and DIS3-AID cells treated or not treated with auxin. These show upregulation of short 3' extended versions of each (boxed) in auxin-treated EXOSC10-AID cells. y axis units are RPKM.

Fig 5: 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 a-GFP (to detect GFP-MTR4), a-EXOSC10, or a-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.