Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents may be directed to and will be fulfilled by the Lead Contact Christopher D. Lima ( limac@mskcc.org ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

E. cloni 10G competent bacterial cells were used for molecular cloning of exosome expression plasmids. Protein subunits were expressed in E. coli BL21CodonPlus (DE3)-RIL competent cells, E. coli One Shot BL21 (DE3) STAR chemically competent cells, or E. coli SoluBL21 (DE3) chemically competent cells. Standard culture was performed as follows: Cells were grown with appropriate antibiotics to OD 600 1.0 to 1.8 in Superbroth medium at 37°C, supplemented with 100 % ethanol to a final concentration of 2 % and transferred to an ice bath for 30 minutes. Protein expression was induced with 0.25 mM IPTG overnight at 18°C. Protocol variations for individual exosome subunits can be found in method details.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

E. cloni 10G competent bacterial cells were used for molecular cloning of exosome expression plasmids. Protein subunits were expressed in E. coli BL21CodonPlus (DE3)-RIL competent cells, E. coli One Shot BL21 (DE3) STAR chemically competent cells, or E. coli SoluBL21 (DE3) chemically competent cells. Standard culture was performed as follows: Cells were grown with appropriate antibiotics to OD 600 1.0 to 1.8 in Superbroth medium at 37°C, supplemented with 100 % ethanol to a final concentration of 2 % and transferred to an ice bath for 30 minutes. Protein expression was induced with 0.25 mM IPTG overnight at 18°C. Protocol variations for individual exosome subunits can be found in method details.

METHOD DETAILS Exosome Purification and Complex Reconstitution S. cerevisiae RNA exosomes. Subunits were expressed, purified, and reconstituted into complexes as described previously ( Greimann and Lima, 2008 ; Wasmuth et al., 2017 ). Where indicated, Rrp6 contains a D238N mutation and Dis3 contains both D171N and D551N mutations to render them catalytically inactive. S. pombe RNA exosomes. All DNA sequences for Schizosaccharomyces pombe were obtained from PomBase S . pombe (Database issue: D695–9) genome data ( Wood et al., 2012 ). cDNA sequences for S . pombe genes rrp41, rrp42, rrp43, rrp45, rrp46, mtr3, rrp4, rrp40, csl4, rrp6 , and dis3 were codon-optimized, synthesized for expression in E. coli , and inserted into sub-cloning vectors (DNA 2.0/ATUM, Newark, CA). Additional codon-optimized cDNA sequences rrp47/C1D, mpp6 , and mtr4 were obtained from IDT DNA and cloned using pGEM vectors. Expression of S . pombe proteins was achieved by sub-cloning DNA sequences into the MCS1 and MCS2 of plasmid pRSF-SMT3-Duet1 (Novagen) vector by use of restriction sites engineered into positions flanking the coding sequence. S . pombe rrp41 and rrp45 cDNA were inserted into the MCS1 and MCS2, respectively. S . pombe rrp42 and mtr3 cDNA were inserted into the MCS1 and MCS2, respectively. S . pombe rrp43 and rrp46 cDNA were inserted into the MCS1 and MCS2, respectively . S . pombe rrp6 and rrp47 cDNA were inserted into the MCS1 and MCS2, respectively. S . pombe csl4, rrp4, rrp40, dis3, mpp6 , and mtr4 were each cloned separately solely into MCS1. Additional point mutations were produced by QuikChange mutagenesis (Agilent Technologies) and sequenced for fidelity to render Rrp6 and Dis3 catalytically inert: the exoribonucleolytic mutation for S . pombe Rrp6 was D243N and the endo- and exoribonucleolytic mutations for S . pombe Dis3 were D166N and D516N, respectively. All wild-type and mutant protein expression plasmids were transformed into One Shot BL21 Star (DE3) (Thermo Fisher Scientific) for protein expression. For expression, cells were grown in shaker flasks to 1.0 OD 600 , and induced overnight at 18°C with 0.25 mM IPTG in the presence of 2% ethanol. After induction, cells were harvested and flash-frozen in liquid nitrogen. Individual S . pombe protein subunits (spCsl4, spRrp4, spRrp40, spDis3, spMtr4, and spMpp6) and heterodimers (spRrp41/Rrp45, spRrp42/Mtr3, spRrp43/Rrp46, spRrp6/Rrp47) were each purified separately. To prepare cell lysate for protein purification, sonication was performed using a cell disrupter in 50 mM Tris-HCl pH 8.0, 20% (w/v) sucrose, 350 mM NaCl, 20 mM imidazole, 0.1% IGEPAL, 1 mM PMSF, 1 mM β-mercaptoethanol (BME), and 10 ug/mL DNase on the thawed cells. Lysate was then centrifuged using a Beckman JA-20 rotor at 44,000× g . Supernatant was applied to Ni-NTA resin (QIAGEN) for purification, and each of the His-tagged Smt3 fusion proteins and Smt3-fusion protein heterodimer pairs were eluted in 20 mM Tris-HCl pH 8.0, 350 mM NaCl, 250 mM imidazole, and 1 mM BME. For further purification, Smt3 fusion proteins were cleaved by Ulp1 overnight at 4°C and fractionated by size exclusion chromatography for spCsl4, spRrp4, spRrp40 with a HiLoad Superdex 75 PG 26/60 (GE Healthcare) and for spRrp41/Rrp45, spRrp42/Mtr3, spRrp43/Rrp46, spRrp6/Rrp47, spDis3, spMtr4, and spMpp6 on a HiLoad Superdex 200 PG 26/60 (GE Healthcare) in Sizing Buffer: 20 mM Tris-HCl pH 8.0, 350 mM NaCl, and 1 mM BME. For each protein subunit or heterodimer sample, peak fractions were pooled, concentrated to 5–10 mg/mL, and flash-frozen prior to reconstitution. S . pombe complexes were reconstituted in three steps: reconstitution of the spExo9 core, reconstitution of the core with the exoribonucleolytic subunits and co-factors (spRrp6/Rrp47, spDis3, spMpp6) to form spExo13, and reconstitution of the spExo14 by addition of the helicase Mtr4 to spExo13. To form the spExo9 core, each of S . pombe core subunits (spCsl4, spRrp4, spRrp40, spRrp41/Rrp45, spRrp42/Mtr3, spRrp43/Rrp46) were added together at equimolar concentrations. The mixture was then dialyzed overnight at 4°C to 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM BME. The dialysate was separated from free subunits using a HiLoad Superdex 200 PG 26/60 (GE Healthcare) in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1 mM BME, and then peak fractions were further purified with a linear salt gradient via a Mono Q 10/100 GL column (GE Healthcare) with Buffer A: 20 mM Tris-HCl pH 8.0, 100mM NaCl, and 1 mM BME and Buffer B: 20 mM Tris-HCl pH 8.0, 1 M NaCl, and 1 mM BME. To reconstitute spExo13, the resulting spExo9 core was mixed with 1.5-fold molar excess of purified spRrp6/Rrp47 and spDis3 and 3-fold molar excess of spMpp6. Mixtures were then dialyzed overnight at 4°C to 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM BME. The dialysate was then applied to a HiLoad Superdex 200 PG 26/60 (GE Healthcare) in 20 mM Tris-HCl pH 8.0, 100mM NaCl, and 1 mM BME to separate away free enzymes from the reconstituted complex. Peak fractions containing stoichiometric quantities of all subunits of spExo13 were pooled and added to a 1.5-fold molar excess of spMtr4 to reconstitute spExo14. Mixtures were then dialyzed in two steps overnight at 4°C to 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM BME. The dialysate was then applied to a Superdex 200 Increase 10/300 GL column (GE Healthcare) in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.5 mM MgCl 2 , 0.5 mM TCEP to remove excess spMtr4. All reconstitutions were concentrated to 7–10 mg/mL by centrifugation in an Amicon Ultra-15 30,000 MWCO filtration unit (Millipore-Sigma), flash frozen in liquid nitrogen, and stored at −80°C. H. sapiens RNA exosomes. RNA exosome core subunits were expressed, purified and reconstituted as described previously ( Greimann and Lima, 2008 ; Wasmuth et al., 2014 ). Full-length human EXOSC10 was codon-optimized for expression in E. coli using the GeneArt platform (Thermo Fisher Scientific) and inserted into a pRSF-His 6 Smt3-Duet vector. To improve expression, constructs were generated to remove aa 805–885 and replace aa 649–704 with the linker sequence SRGSGSGSGSGS to yield EXOSC10 1–804 Δ649–704 . Catalytically inert EXOSC10 was generated by introducing a D313N mutation. hRrp47 (C1D) was generated from a cDNA library (Ambion) and cloned into MCS2 of the pRSF-His 6 Smt3-EXOSC10 vector. Cloning of different pRSF-Duet-Smt3-EXOSC10/C1D constructs was performed using custom primers and Gibson assembly cloning (New England Biolabs). BL21 (DE3) Codon Plus RIL STAR cells were transformed with the EXOSC10/C1D heterodimer construct and induction, lysis and Ni-affinity purification were performed as described for human exosome core components, however, Ulp1 cleavage of the Smt3 tag was not performed, instead Smt3- EXOSC10/C1D was purified by size exclusion using a HiLoad Superdex 200 PG 26/60 (GE Healthcare) column equilibrated with gel filtration buffer (20 mM Tris-HCl pH 8.0, 350 mM NaCl, 1 mM BME). Peak heterodimer-containing fractions were dialyzed overnight against buffer containing 20 mM Tris-HCl pH 8.0, 250 mM NaCI, and 2 mM TCEP and purified using Heparin HiTrap HP 5 ml affinity columns (GE Healthcare, Buffer A: 20 mM Tris-HCl pH 8.0, 250 mM NaCl, 2 mM TCEP; Buffer B: 20 mM Tris-HCl pH 8.0, 1 M NaCl, 2 mM TCEP, 0–40% buffer B over 20 CV). hDIS3 was generated from a cDNA library and cloned into pGEX-4T2 using SmaI and NotI restriction sites to generate a GST fusion. Inactivating mutations to the endonucleolytic and exonucleolytic sites (D146N and D487N, respectively) were introduced by QuikChange mutagenesis (Agilent Technologies). Expression and induction was performed following the established protocol for the human Exo9 core. For lysis, cells were supplemented to a volume of 250 ml with hDIS3 lysis buffer (20 mM Tris pH 8.6, 350 mM NaCl, 1 mM BME, 0.1% v/v IGEPAL, 10 μg/ml DNAse I, 1 mM PMSF) and disrupted by sonication over three times 2 min at 65% intensity with 1 s on, 3 s off intervals using a Branson Digital Sonifier. Cell lysates were cleared by centrifugation at 44,000× g (Beckman JA-20) for 45 minutes at 4°C. For pull-down of GST-hDIS3, 5 ml of Glutathione Sepharose ™ 4B (GE Healthcare) were equilibrated with binding buffer (20 mM Tris pH 8.6, 350 mM NaCl, 1 mM BME), added to the supernatant and incubated for 1 h at 4°C. Protein-bound resin was washed with 40 volumes of binding buffer and GST-DIS3 was eluted after 15 min of incubation in 7.5 ml elution buffer (20 mM Tris pH 8.6, 350 mM NaCl, 15 mM reduced L-glutathione). Thrombin was added at a concentration of 1.5 U/mg hDIS3 followed by overnight incubation at 4°C. Cleavage products were separated by gel filtration using a HiLoad Superdex 200 PG 26/60 (GE Healthcare) column equilibrated with gel filtration buffer (20 mM Tris-HCl pH 8.0, 350 mM NaCl, 1 mM BME). Peak fractions were dialyzed overnight against buffer containing 20 mM Tris-HCl pH 8.0, 50 mM NaCl, and 2 mM TCEP. Sample was then purified using a MonoQ 10/100 GL column (GE healthcare; buffer A: 20 mM Tris-HCl pH 50 mM NaCl, 2 mM TCEP; buffer B: 20 mM Tris-HCl pH 8.0, 1 M NaCl, 2 mM TCEP, 0–40% buffer B over 20 CV). Human M-Phase-Phosphoprotein 6 (MPP6) gene was PCR-amplified from placenta cDNA pool (Ambion) using Pfu Turbo DNA polymerase (Stratagene) and digested PCR products were inserted into pSMT3-TOPO. Expression and purification was performed analogous to what has been described previously for the human core subunits Csl4, Rrp4 and Rrp46 ( Greimann and Lima, 2008 ; Wasmuth et al., 2014 ). Human MTR4 (SKIV2L2, DOB1) was amplified from human kidney cDNA (Invitrogen) and subcloned into pET-28a with an N-terminal His 6 Smt3 tag. Culture and purification were performed as described for the H. sapiens core subunits using a HiLoad SD200 PG 26/60 size exclusion column. Peak fractions were pooled, dialyzed overnight at 4°C against 100 mM NaCl, 20 mM Tris-HCl pH8.0, 2 mM TCEP and purified over a Heparin HiTrap HP 5ml affinity column (GE Healthcare, Buffer A: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM TCEP; Buffer B: 20 mM Tris-HCl pH 8.0, 1 M NaCl, 2 mM TCEP; 0–100% buffer B over 20 CV). All individual subunits were concentrated to ~10 mg/ml using appropriately sized Amicon® centrifugal filter tubes (Merck Millipore Ltd.), flash-frozen and stored at −80°C. For reconstitution of H. sapiens 12-, 13-, and 14-component RNA exosomes, the Exo9 core was mixed with 4-fold molar excess of EXOSC10/C1D, 2-fold molar excess of DIS3, 7.5-fold molar excess of MPP6 and 2-fold molar excess of MTR4 in 20 mM Tris-HCl 8.0, 350 mM NaCl, 2 mM TCEP supplemented with Ulp1 at a mass ratio of 1:1000 relative to EXOSC10. The mixture was dialyzed for 4 h at 4°C in buffer containing 20 mM Tris-HCl 8.0, 100 mM NaCl, 2 mM TCEP, followed by overnight dialysis in 20 mM Tris-HCl 8.0, 50 mM NaCl, 2 mM TCEP, 0.5 mM MgCl 2 . Reconstituted complexes were purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare) and peak fractions containing stoichiometric amounts of all subunits were concentrated to 10 mg/ml using Amicon® UltraCel® 30K MWCO centrifugal filters (Merck Millipore Ltd.), flash-frozen in liquid nitrogen, and stored at −80°C. Reconstitution of 12- and 13-component exosomes was performed analogously. Biochemical Assays Substrate Preparation. Oligonucleotides were synthesized and HPLC-purified by Integrated DNA Technologies (Coralville, IA) and Dharmacon Inc (Lafayette, CO). Lyophilized RNA was suspended in annealing buffer (20 mM Tris-HCl pH 7.0, 100 mM potassium acetate) and stored at −80°C. Duplex substrates were prepared by heating an equimolar mixture of each strand to 95°C followed by cooling to 16°C for at least an hour. Annealed substrates were purified by size exclusion chromatography using Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated with annealing buffer. Oligonucleotides used to prepare helicase and decay substrates include the following 5′ to 3′ sequences (DNA sequences are underlined): Fluorescein-AGC ACC GUA AAG ACG C (RNA displacement strand, Figure 1A,B ), GCG UCU UUA CGG UGC UAA AAA AAA AAA AAA AAA AAA (RNA translocation strand, Figure 1A ), GCG UCU UUA CGG UGC UAA AAA AAA AAA AAA AAA AAA (DNA-RNA chimera translocation strand, Figure 1B ), AGC ACC GUA AAG ACG C (RNA displacement strand, Figure 2C , Figure S1B,C ), GTG TGG TGT GGT GTG GTG TGG TGT GGT GTG GTG TGG T (DNA displacement strand, Figure 2C ) Fluorescein-GCG UCU UUA CGG UGC U CA CCA CAC CAC ACC ACA CCA CAC CAC ACC ACA CCA CAC AAA AAA AA (RNA translocation strand, Figure 2C ), Fluorescein- GCG TCT TTA CGG TGC T CA CCA CAC CAC ACC ACA CCA CAC CAC ACC ACA CCA CAC AAA AAA AA (RNA/DNA translocation strand, Figure S1B ), GCG TCT TTA CGG TGC T CA CCA CAC CAC ACC ACA CCA CAC CAC ACC ACA CCA CAC AAA AAA AA (DNA-RNA translocation strand, Figure S1C ), Fluorescein- GTG TGG TGT GGT GTG GTG TGG TGT GGT GTG GTG TGG T (DNA displacement strand, Figure S1C ). See also Table S2 . Unwinding assays. Assays in main Figure 1A,B were carried out at 22°C in a buffer containing 20 mM Tris-HCl pH 7.0, 50 mM NaCl, 0.5 mM MgCl 2 , 5 mM BME, and 1 U/μl RNAse inhibitor, human placenta (New England Biolabs). Substrates (10 nM) were pre-incubated with 200 nM protein for at least 5 minutes. Reactions were initiated by addition of 2 mM ATP, 2 mM MgCl 2 , and 100 nM DNA trap (5′ GCGTCTTTACGGTGCT 3′). Aliquots were taken at indicated times and quenched using 0.25% v/v SDS, 5 mM EDTA, 10% v/v glycerol and 80 mU/μl Proteinase K (New England Biolabs). Samples were resolved in Novex 20% TBE gels (Thermo Fisher Scientific), imaged using Typhoon FLA 9500 laser scanner (GE Healthcare) and analyzed using ImageJ ( Schneider et al., 2012 ). For helicase time course assays in Figure S1C . S . cerevisiae . Final concentrations were 20 nM exosome, 22 nM Mtr4, 1 mM ATP or AMPPNP, 0.5 UμL RNAse inhibitor (New England Biolabs), and 10 nM RNA substrate in RNA decay buffer. Exosome complexes or buffer was incubated on ice with Mtr4 at 2 μM and 2.2 μM, respectively, in RNA decay buffer for 1 hour prior to initiating the reaction. A mix containing RNA and RNAse inhibitor (all at 1.25× final concentration) was incubated at 20°C for 5 min prior to addition of 1/10 volume of 200 nM enzyme. The reaction was then initiated with 1/10 volume of an initiation mix containing 10 mM ATP or AMPPNP and 300 nM DNA trap oligo (sequence = 5′ ACACCACACCACACCAC 3′). Reactions were quenched after the indicated incubation times by adding 10 μL of reaction to 5 μL of stop mix (0.3 % w/v SDS, 30 mM EDTA pH 8.0, 3 U/mL proteinase K (New England Biolabs)) followed by proteinase K digestion at 37°C for 1 hour and flash freezing in liquid nitrogen for storage at −80°C. 3.5 μL sample was loaded per lane and run for 45 minutes on 4–20% polyacrylamide-TBE gels (Life Technologies) using 0.5× TBE as running buffer. Gels were imaged on Typhoon FLA 9500 laser scanner (GE Healthcare) using the FAM setting at 600V and analyzed using ImageJ ( Schneider et al., 2012 ). H. sapiens . Assays were performed as described for S . cerevisiae using reconstituted complexes. Quenching buffer was modified to contain 80 U/mL Proteinase K with overnight digestion at 30 °C. S . pombe . Assays were performed as described for S . cerevisiae . Site-directed 4-thiouridine UV crosslinking assay. Protein samples (200 nM) were incubated with 100 nM 5′ fluorescein- and internal 4SU-labeled RNA in a buffer containing 20 mM Tris-HCl pH 7.0, 100 mM NaCl, 2.5 mM MgCl, 5 mM BME, and 2 mM AMPPNP or ATP. To induce crosslinking, samples were exposed to long-range UV (365 nm) for 20 minutes at 4°C using a 4W handheld UV lamp (UVP). RNA-protein adducts were separated using NuPage 4–12% Bis-Tris protein gels (Thermo Fisher Scientific), visualized using Typhoon FLA 9500 laser scanner (GE Healthcare) and analyzed using ImageJ ( Schneider et al., 2012 ). Oligonucleotides used to prepare substrates include the following 5′ to 3′ sequences of the translocation strands: Fluorescein- GCG TCT TTA CGG TGC T AA AAA AAA AAA AAA AAA A(4SU)A, Fluorescein- GCG TCT TTA CGG TGC T AA AAA AAA AA(4SU) AAA AAA AAA, Fluorescein- GCG TCT TTA CGG TGC T AA AAA (4SU)AA AAA AAA AAA AAA, Fluorescein- GCG TCT TTA CGG TGCT (4SU)A AAA AAA AAA AAA AAA AAA, Fluorescein- GCG TCT TTA CGG TGC T CAC ACC ACA CCA CAC CAC ACA AAA AA(4SU) A, Fluorescein- GCG TCT TTA CGG TGC T CAC CAC ACC ACA CCA CAC CAC ACC ACA CCA CAC CAC ACA AAA AA(4-S-U) A; RNA top strand: AGC ACC GUA AAG ACG C; and DNA displacement strands: TTT TTT TTT TT , TTA TTT TTT TT, TTT TTT ATT TT , GTG TGG TGT GGT GTG GT GT , GTG TGG TGT GGT GTG GTG TGG TGT GGT GTG GTG TGG T ; DNA sequences are underlined. See also Table S2 . RNA decay assays. S. cerevisiae. For the decay time courses in Figure 2C and Figure S1B , final concentrations were 20 nM exosome, 22 nM or 0 nM Mtr4, 1 mM ATP or AMPPNP, 0.5 U/μL RNAse inhibitor (New England Biolabs), and 10 nM RNA substrate in RNA decay buffer (20 mM HEPES-KOH pH 7.5, 50 mM potassium acetate, 1.1 mM magnesium acetate, 2.5 mM DTT, 0.01 % IGEPAL). 50 mM ATP and AMPPNP stock solutions were adjusted to pH 7.0 with KOH. Exosome complexes were incubated on ice with Mtr4 (or buffer) at 2 μM and 2.2 μM, respectively, in RNA decay buffer for 1 hour prior to diluting 1:10 in RNA decay buffer and initiating the reaction. A mix containing RNA, ATP or AMP-PNP, and RNAse inhibitor (all at 1.1× final concentration) was incubated at 20°C for 5 min prior to initiation with 1/10 volume of 200 nM enzyme mix. Reactions were quenched after the indicated incubation times by adding 10 μL of reaction to 5 μL of stop mix (0.3 % w/v SDS, 30 mM EDTA pH 8.0, 3 U/mL proteinase K (New England Biolabs) followed by proteinase K digestion at 37°C for 1 hour and flash freezing in liquid nitrogen for storage at −80°C. For native gels, 3.5 μl sample was loaded per lane and run for 45 minutes on 4–20% acrylamide-TBE gels using 0.5× TBE as running buffer. Gels were imaged on a typhoon FLA 9500 instrument (GE Healthcare) using the FAM setting at 600V and analyzed using ImageJ ( Schneider et al., 2012 ). H. sapiens. Assays were performed as described for S . cerevisiae but using reconstituted complexes at a final concentration of 100 nM. Quenching buffer was modified to contain 80 U/ml Proteinase K (New England Biolabs) and digestion was carried out at 30°C over night. S. pombe. Assays were performed as described for S. cerevisiae but using reconstituted complexes at a final concentration of 100 nM for Exo13 and Exo14. For exosomes lacking Rrp6/Rrp47, Exo10 Dis3 was added to Mpp6 and Mtr4.

Electron Microscopy Sample Preparation and Imaging

Substrate-loading hExo14 for cryo-electron microscopy. Large-scale loading reactions of human Exo14 were performed in a total volume of 100 μl (20 mM Tris pH 8.0, 50 mM NaCl, 0.5 mM MgCl 2 , 2.5 mM BME) at 4.5 μM hExo14 with stoichiometric amounts of substrate. DNA-RNA/DNA substrate and complex were mixed and incubated on ice for 15 min and loading was initiated by adding 10 μl of 20 mM Mg·ATP, 45 μM trap and incubated at 22°C for 2 h. 50 μl of 20 mM Tris pH 8.0, 50 mM NaCl, 6 mM AMPPNP, 2.5 mM MgCl 2 , 1 mM BME was added to quench the reaction, cleared by high-speed centrifugation and purified using a Superdex 200 Increase 10/300 GL column (GE Healthcare) in 20 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM MgCl 2 , 1 mM BME. Peak fractions were supplemented with quenching buffer to achieve a final concentration of 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2.5 mM MgCl 2 , 2 mM AMPPNP, 1 mM BME. Sample was concentrated to >1 mg/ml using Amicon® UltraCel® 30K MWCO centrifugal filters (Merck Millipore Ltd.), supplemented with 0.05% CHAPSO and immediately used for grid preparation. Aliquots for analysis of nucleic acid were added to loading buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2.5 mM BME, 0.5 mM MgCl 2 , 20% v/v glycerol, 10 mM EDTA, 0.2 % v/v SDS, 50 U/ml Proteinase K) and incubated at 37°C for 3 h. Aliquots for analysis of protein were added to 4× LDS sample buffer supplemented with 5% v/v BME and separated by SDS-PAGE followed by Coomassie or SYPRO Ruby staining. Cryo electron microscopy data collection. Quantifoil® R 2/2 gold grids were glow-discharged for 60 sec at 0.37 mBar, 15 mA in a Pelco easiGlow™ air system (TED PELLA, Inc.). Four μl of a suspension of RNA-loaded hExo14 complexes at a concentration of 1.75 μM protein were applied per grid and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI) at 20°C, 100% humidity (30 sec wait time, 2.5 sec blot time). Grids were screened for suitable particle density using an MSKCC in-house Titan Krios 300KV (FEI) microscope and Serial EM data collection software ( Mastronarde, 2005 ). Data collection was performed on a Titan Krios 300KV (FEI) instrument using a K2 Summit direct detector (Gatan, Inc.) and Leginon data acquisition interface ( Suloway et al., 2005 ). A total of 1604 movies (50 frames/movie, 10 sec exposure time) were collected in super-resolution mode with a defocus range from −1.0 to −3.0 μm at a dose rate of 10.0 e-/px/sec and a total dose of 85.23 e-/Å 2 /movie. The exposure of the movies was filtered in a dose-dependent manner to weight the amplitudes of each movie frame to preserve low resolution contrast across the exposure while favoring high resolution information from early frames that sustained less radiation damage, as described ( Grant and Grigorieff, 2015 ). The calibrated pixel size was 1.07 Å/px.

Image Processing and Map Reconstruction

Structure determination. Movies were processed with MotionCor2 to generate dose-weighted aligned and averaged micrographs alongside aligned movies ( Zheng et al., 2017 ). 1439 micrographs were selected for further processing based on their appearance, CTF estimation, and number of particles. In RELION ( Scheres, 2012 ), a few hundred particles were selected to generate a small set of 2D classes that were then used as templates for autopicking the remaining particles. This particle stack was input to cryoSPARC ( Punjani et al., 2017 ) to obtain an initial set of 2D class averages and to remove junk particles or particles that could not be classified. This procedure resulted in 278185 particles whose coordinates were used in RELION for movie processing and particle polishing. After another round of 2D classification in RELION, the resulting stack containing 271036 particles was then used in cryoSPARC to generate four ab initio 3D reconstructions, one for the helicase exosome complex (122703 particles), one for the core and DIS3 (64191 particles), and one for the core (49326 particles). The latter two include diffuse densities at the top of the core that may be EXOSC10 or nucleic acid. The remaining particles contributed to a fourth reconstruction that appears to encompass partial complexes of the Exo9 core. Particles associated with the first three models were combined and used as input to cryoSPARC for heterogeneous refinement followed by homogeneous refinement. Further inspection of the helicase complex revealed well-resolved densities for the nine-subunit exosome core, most of MPP6, a peptide of EXOSC10 bound to the exosome core, and lower resolution densities for the helicase, nucleic acid and DIS3. The 122703 particles for the helicase complex were then transferred to RELION for an independent round of 2D classification that resulted in 122284 particles that were input to 3D refinement, generating a map that closely resembled the map obtained from cryoSPARC (correlation coefficient = 0.985). The resolution of this map was assessed to be 3.45 Å (or 3.55 Å) depending on whether the half-maps were masked to exclude (or include) the disordered KOW domain of MTR4. All map resolutions throughout were assessed using FSC of 0.143. To better resolve densities for the core, MTR4 and DIS3, aligned particles were used as input for focused 3D classification or focused 3D refinement. For the core, 3D classification did not reveal any major subclasses, however focused 3D refinement improved densities for the core (RELION 3.31 Å; phenix.mtriage 3.23 Å) at the expense of densities for MTR4 and DIS3. Focused refinement also resulted in an improved map for MTR4 (RELION 3.54 Å; phenix.mtriage 3.45 Å), although densities for duplex DNA/RNA portion of the substrate or the KOW domain remained poorly resolved. Densities for the DIS3 RNB and CSD domains could not be improved through focused refinement using all of the particles, but 3D classification revealed a major class (91483 particles) that was used in focused 3D refinement encompassing all of DIS3, followed by focused refinement with local searches with each of the three individual segments, the DIS3 PIN, cold-shock domains (CSDs), and the ribonuclease domain (RNB). While focused refinement on the RNB improved densities, focused refinement on the CSDs or PIN did not, presumably due to limited signal in these regions. Densities for the CSDs and RNB were improved by combining both regions in focused refinement (RELION 3.90 Å; phenix.mtriage 3.58 Å) while densities for the PIN were improved by focused refinement using a mask that encompassed the PIN domain and three proximal PH-ring subunits of the core (RELION 3.40 Å; phenix.mtriage 3.57 Å). To better resolve densities for the duplex region of the DNA/RNA substrate and KOW domain, focused 3D classification of MTR4 was conducted in two rounds, one without resolution limits, and a second round that limited resolution to 6 Å that resulted in several subclasses, some of which were refined. In the first round, two small subclasses (3643 and 2973 particles) revealed densities for the KOW domain after refinement at estimated resolutions 7.75 Å and 8.22 Å, respectively (RELION). The second round of 3D classification resulted in a class with resolved densities for the duplex region of our substrate. This class was subjected to focused refinement as described above for MTR4 and resulted in an improved map (RELION 3.90 Å; phenix.mtriage 3.82 Å).

Model Building and Refinement

The model for the human Exo9 nine-subunit core (PDB: 2NN6) was used alongside models for the yeast eleven and twelve-subunit exosome bound to Rrp6 and Mpp6 (PDB: 5K36 and 5VZJ) to manually inspect and rebuild the human exosome core using Coot ( Emsley et al., 2010 ). MPP6, EXOSC10 and DIS3 were manually rebuilt based on electron densities using models based on similarities to available yeast counterparts or mouse DIS3L2 (PDB: 4PMW). The MTR4 core was rebuilt after docking a high-resolution x-ray structure of human MTR4 (PDBID: 6C90) into density. A portion of the two helical stalks that bridge the helicase and KOW domains were included based on homology to the yeast enzyme, and densities from the two lower-resolution maps with visible KOW domains. Models were fit into density and refined in real space using Coot ( Emsley et al., 2010 ) (RRID:SCR_014222). Resulting models were refined in Phenix using real-space with ADP refinement ( Adams et al., 2010 ) (RRID:SCR_014224) against individual maps obtained from focused 3D refinement. A final composite model was generated (PDB: 6D6R). This model was refined against the overall map using the starting model as a restraint with ADP refinement to generate a refined model in the overall reconstruction (PDB: 6D6Q). Figures depicting structures and maps were prepared using PyMol ( Schrödinger, 2015 ) (RRID:SCR_000305) or Chimera ( Pettersen et al., 2004 ). Structure quality was assessed using MolProbity ( Chen et al., 2010 ) (RRID:SCR_014226).

Supplementary Material 1 Figure S1. Nomenclature, helicase-dependent degradation and unwinding of the DNA-RNA chimera, and purification of a substrate-loaded human RNA exosome, Related to Figures 1 and 2 . (A) Nomenclature of exosome subunits and co-factors from Homo sapiens , Saccharomyces cerevisiae , and Schizosaccharomyces pombe . (B) Time-course assays for degradation of the DNA-RNA chimera in the presence of AMPPNP (left) or ATP (right) (similar to Figure 2C ). The substrate used is schematically represented at the top; cartoons are labeled to represent subunit compositions. (C) ATP-dependent unwinding of the DNA-RNA chimera by 12-, 13- and 14-component exosomes as well as Mtr4. Marker (M) for single stranded species with sizes indicated to the right and marker (M2) for the displaced fluorescein-labelled strand with trap oligo. Gel images in panels B and C are representative of two independent experiment for S . cerevisiae and three independent experiments for S. pombe and human. (D) Sybr Gold-stained gels of proteinase K-digested fractions after RNA-unwinding and size exclusion chromatography. Top: Fractions 17–22 with corresponding RNA markers (MS=substrate; MP=loaded product) at indicated concentrations. Bottom: Fractions 24–29 with corresponding RNA markers. Band intensity does not scale between individual gels. * denotes peak fractions containing unwound reaction product. (E) Coomassie stained SDS-PAGE protein fractions corresponding to panel D. Gel images in panels D and E are representative of two independent experiments. Load = RNA unwinding reactions prior to gel filtration; M = molecular weight marker. 2 Figure S2. Schematic depicting the overall single particle analysis used to obtain electron density maps, Related to Figure 3 . Boxed maps (green) with labels represent endpoints for those maps available for download in EMDB. 3 Figure S3. Particle angular distribution and reconstructions with local resolution estimates, Related to Figure 3 . (A) Schematic representing the angular distribution of particles contributing to the overall reconstruction as reported by RELION and displayed using Chimera with the overall reconstruction rendered as isosurface inside the sphere in four orientations. (B,C) Overall reconstruction and gold-standard FSC. (D,E) Reconstruction after focused refinement on the core and gold-standard FSC. (F,G) Reconstruction after focused refinement on the DIS3-PIN and gold-standard FSC. (H,I) Reconstruction after focused refinement on the DIS3-RNBCS and gold-standard FSC. (J,K) Reconstruction after focused refinement on MTR4 and gold-standard FSC. (L,M) Reconstruction after focused refinement on a subclass of MTR4 particles elicited more continuous densities for the duplex DNA/RNA segment of the substrate.The six reconstructions analyzed in this study (and boxed in Figure S2 ) are isosurface rendered and colored according to local resolution as calculated by RESMAP ( Kucukelbir et al., 2014 ). Separate from this calculation, the maps were low-pass filtered at 4.5 Å resolution to enable visualization of the most disordered regions at low contour threshold (left), at higher contour threshold (middle), and as a central slice (right) in panels B, D, F, H, J, and L. Color code for resolution shown adjacent to each map. Gold-standard FSC curves corresponding to each reconstruction with FSC=0.143 represented as a dashed line and resolutions indicated are shown at far right. 4 Figure S4. Stereo views of representative electron densities before and after focused refinement, Related to Figure 3 . Labels indicated for each map as labeled in Figure S2 and S3 . Panels showing regions around: (A) MTR4 in the overall map. (B) MTR4 in the MTR4-focused map. (C) MTR4 the MTR4-dsDNA/RNA focused map. (D) Regions within the core for the overall map (E) Regions within the core for the core focused map. (F) Densities surrounding the PIN domain of DIS3 for the overall map (G) Densities surrounding the PIN domain of DIS3 for the DIS3 PIN focused map. (H) Densities surrounding the DIS3 RNB and cold-shock domains for the overall map. (I) Densities surrounding the DIS3 RNB and cold-shock domains for the DIS3 RNBCS focused map. 5 Figure S5. 2D class averages, micrographs, model validation, and FSC curves, Related to Figures 3 and 5 . (A) 2D class averages obtained from cryoSPARC calculated using all particles selected for the final 3D reconstruction. (B) A representative micrograph from the dataset used to determine the structure showing particles (left) and with green circles depicted around selected particles (right). Scale bar at lower right (30 nm). (C) Gold-standard FSC between the respective coordinate models for the overall refinement and local regions of the complex as indicated as calculated by phenix.mtriage. FSC values at 0.5 and 0.143 are reported in panel A. The DIS3 PIN focused refinement utilized a mask covering the PIN and most of the three adjacent PH-like proteins, however the gold-standard FSC between the DIS3 PIN model and the corresponding focused refinement map overestimated the resolution of the DIS3 PIN if core subunits were included. Therefore, the DIS3 PIN was compared to a modified version of the focused refinement map whereby densities attributed to the core subunits were removed. (D) Gold-standard FSC curve (FSC=0.143, dotted line) from RELION reconstruction shown in Figure 5G . (E) Gold-standard FSC curve (FSC=0.143, dotted line) from RELION reconstruction shown in Figure 5H . 6 Figure S6. EXOSC10 peptide located between EXOSC6 and EXOSC8 and interactions between DIS3 and the exosome core, Related to Figure 3 . (A) Structural representation of contacts between EXOSC10 and EXOSC6 as well as EXOSC8 colored as in Figure 3 . (B) Stereo view of electron densities overlaying the corresponding areas shown in panel A with blue mesh for EXOSC10 and grey mesh for EXOSC6 and EXOSC8. (C) View of DIS3 contacts to the core with boxes representing regions indicated in detail in panels D-I. Subunits, domains and RNA are labeled and colored as in Figure 3 . (D) Interactions between EXOSC9 and the DIS3 RNB domain. (E) Interactions between EXOSC9 and the DIS3 CSD2 domain. (F) Interactions between the DIS3 CSD1 domain and EXOSC7 and EXOSC8. (G) Interactions between the DIS3 CSD2 domain and EXOSC9 and EXOSC4. (H) Interactions between EXOSC4 and the DIS3 PIN domain. (I) Interactions between the DIS3 PIN domain and EXOSC4 and EXOSC7.Side chains shown in stick representation, labeled by residue number (colored by subunit), polar interactions indicated by dashed lines between atoms, and domains indicated by labels. 7 Figure S7. Interactions between RNA and EXOSC4/EXOSC9, comparison to the RNA binding site in a phosphorolytic enzyme, comparison of RNA paths in other Dis3 structures, and structure based sequence alignments indicating subunit contacts, Related to Figures 4 , 6 , and 7 . (A) RNA contacts at a conserved interface between EXOSC9 and EXOSC4. (B) RNA contacts within the active site between Rrp41/Rrp42 in the phosphorolytic archaeal S . solfataricus exosome (PDBID: 2C37). Structures were aligned with protein and RNA shown in stick representation with amino acids colored and labeled and bases in the human complex labeled.

RNA bound Dis3 structures shown to depict different

RNA paths for (C) Human DIS3. (D) Mouse DIS3L2 (PDBID: 4PMW). (E) S . cerevisiae Dis3 from a core bound structure in the ‘open’ conformation (PDBID: 5K36). (F) S . cerevisiae Dis3 from a core bound structure in the ‘closed’ conformation (PDBID: 4IFD). RNB domains were used to align the respective Dis3 structures with Dis3 domains colored as CSD1 (orange), CSD2 (limon), RNB (pink), and S1 (purple). RNA in respective structures is depicted as a blue tube to highlight the different binding modes. The PIN domains for human DIS3 and budding yeast structures are omitted for clarity. (G) Amino acid sequence alignment of Mtr4 from various species. Alignment generated using Muscle alignment tool in Jalview ( Waterhouse et al., 2009 ) with conservation based on BLOSUM62 scoring matrix. EXOSC2 residues that interact with MTR4 in this structure are indicated by green bars above the sequence. (H) Alignment of EXOSC2 and Rrp4 proteins generated using MUSCLE multiple sequence alignment tool. Conservation based on BLOSUM62 scoring in Jalview. Colored bars indicate surfaces involved in protein-protein interactions based on assessment of the human structure (this work) and a previously published S . cerevisiae structure ( Zinder et al., 2016 )(PDBID: 5K36). (I) Sequence alignment of MPP6 from indicated organisms. Alignment generated using MUSCLE alignment tool and manually corrected for S . cerevisiae and H. sapiens based on alignment of the human structure to a published structure from yeast (PDBID: 5VZJ)( Wasmuth et al., 2017 ). Conservation indicated by shading and based on BLOSUM62 scoring in Jalview ( Waterhouse et al., 2009 ). Tick marks correspond to 10 amino acid intervals for the human MPP6 sequence. Colored bars indicate residues involved in protein-protein interactions with MTR4, EXOSC1 and EXOSC3 (or Rrp40) with dark shading indicating direct contacts based on PDBe PISA (cutoff for buried area 50%) and further visual assessment of the corresponding density for the human structure. Dark gray lines above or below the sequence alignment indicate residues modeled in this work or previous work in the yeast system, respectively. C-terminal amino acids from all species were omitted for clarity. 8