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. Author manuscript; available in PMC: 2021 Nov 17.
Published in final edited form as: Methods Mol Biol. 2020;2062:449–465. doi: 10.1007/978-1-4939-9822-7_22

Reconstitution of the Schizosaccharomyces pombe RNA exosome

Kurt Januszyk 1, Christopher D Lima 1,2
PMCID: PMC8596990  NIHMSID: NIHMS1750569  PMID: 31768990

Abstract

In this chapter, we describe methods to clone, express, purify, and reconstitute active S. pombe RNA exosomes. Reconstitution procedures are similar to methods that have been successful for the human and budding yeast exosome systems using protein subunits purified from the recombinant host E. coli. By applying these strategies, we can successfully reconstitute the S. pombe non-catalytic exosome core as well as complexes that contain the exoribonucleases Dis3 and Rrp6, cofactors Cti1 (equivalent to budding yeast Rrp47) and Mpp6 as well as the RNA helicase Mtr4.

Keywords: RNA exosome, RNA decay, Ribonuclease, Helicase, fission yeast

1. Introduction

The RNA exosome is a ubiquitous exoribonuclease in eukaryotic cells that coordinates with multiple cofactors for processing and degradation of virtually all classes of RNA [1–3]. The eukaryotic RNA exosome has been extensively studied in the budding yeast system, to a lesser extent in the human system, and even less so in fission yeast. While many facets of RNA biology can be extrapolated to higher eukaryotes from budding yeast, the RNA exosome plays an important role in many pathways (e. g. RNA interference biology and chromatin modification) that are absent or more primitive in budding yeast [4]. From this standpoint, we sought to utilize the S. pombe system for studying RNA exosome biology that bridges the functions associated with protozoan and metazoan biology. In fact, the Grewal, Moazed, and Bachand groups (among others) have specifically studied the RNA exosome and associated cofactors within S. pombe cells [5–9].

The S. pombe nuclear RNA exosome also consists of the nine-subunit core (Exo9), the processive exoribonuclease Dis3, and the distributive exoribonuclease Rrp6 [10–12]. The nuclear exosome complex interacts with an array of cofactors to process or degrade different RNA substrates, including the cofactors Cti1/Rrp47/C1D and Mpp6 [13–16]. In S. pombe, these two cofactors are also believed to facilitate the interaction with the DExH helicase Mtr4, similar to the equivalent proteins in the budding yeast system. S. pombe Mtr4 has been detected in the TRAMP complex (which contains the Mtr4 helicase, Trf4/Trf5/Cid14 poly(A) polymerases, and Air1/Air2 Zn-knuckle RNA binding proteins) to promote surveillance and degradation of aberrant RNA [17]. However, there are features that may turn out to be unique to S. pombe or not discovered yet in higher eukaryotes; for instance, the S. pombe system contains two orthologous Mtr4 proteins Mtr4 and Mtl-1 (Mtr4-like protein 1), the latter has been shown to be a member of the MTREC/NURS complex that is involved in targeting meiotic RNAs [11,12]. For these reasons, it is imperative to have a reconstituted system to characterize the activities associated with S. pombe RNA exosomes. Protocols will be described in this section for the purification and reconstitution of the S. pombe nuclear RNA exosome that have been successfully implemented for biochemical characterization of this complex [18].

2. Materials

2.1. Cloning

  1. Restriction Enzymes.

  2. Quickchange Site-Directed Mutagenesis Kit.

  3. Standard DNA Electrophoresis Systems.

  4. Vectors: pRSF-DUET-His6-Smt3 (modified Novagen vector), pGEM (Promega).

2.2. Protein Expression and Purification

  1. ELGA Purelab Ultra 18.2 MΩ-cm ultrapure water or equivalent for all solutions.

  2. 250 mL and 50 mL conical tubes, 1.5 mL microcentrifuge tubes.

  3. Refrigerated microcentrifuge capable of 20,000 x g force.

  4. Luria-Bertani (LB) and LB-agar pellets, Super Broth (SB) pellets.

  5. Antibiotics: 50 mg/ml kanamycin in water (0.2 μM filtered); 50 mg/ml ampicillin in water (0.2 μM filtered).

  6. Bacterial strains for protein expression: E. coli BL21 One Shot STAR (DE3) (Stratagene) for protein expression and E. coli One Shot TOP10 cells (Thermo Fisher) for cloning.

  7. Shaking incubators (18-37 °C) and 2 L baffled shaking flasks.

  8. Antifoam 204.

  9. Beta-mercaptoethanol (BME).

  10. Ulp1 protease (Invitrogen or purified in-house) [19].

  11. 1 M Tris-HCl (pH 8.0 at 20°C).

  12. 5 M NaCl.

  13. 0.25 M Isopropyl β-D-thiogalactopyranoside (IPTG).

  14. 100 mM phenylmethylsulfonyl fluoride (PMSF) in aqueous isopropanol.

  15. 1.0 M MgCl2.

  16. 2.5 M imidazole (adjusted to pH 8.0 at 20°C with concentrated HCl).

  17. 10 % (v/v) IGEPAL ca-630.

  18. Stock solutions of 10 mg/mL lysozyme and DNAse I in 20 mM Tris-HCl (pH 8.0), 50 mM NaCl aliquoted, flash frozen and stored at −20°C.

  19. Tris-sucrose buffer: 50 mM Tris-HCl pH 8.0, 20% (w/v) sucrose (stored at 4°C).

  20. Branson 450 watt digital sonifer equipped with ½ inch disrupter horn and acoustic enclosure.

  21. Beckman Coulter Avanti J-26XP (or similar) equipped with JLA 8.1 and JA-20 rotors.

  22. Buffer T350: 20 mM Tris-HCl pH 8.0, 350 mM NaCl, 1 mM BME (stored at 4°C).

  23. Ni wash buffer: 20 mM Tris-HCl pH 8.0, 350 mM NaCl, 10 mM imidazole, 1 mM BME (stored at 4°C).

  24. Ni elution buffer: 20 mM Tris-HCl pH 8.0, 350 mM NaCl, 250 mM imidazole, 1 mM BME (stored at 4°C).

  25. Ni-NTA agarose resin.

  26. Empty, reusable columns.

  27. AKTA-FPLC (GE Healthcare) equipped with 10 mL, 1.0 mL, 0.5 mL and 0.4 mL injection loops, gel filtration columns (HiLoad 26/60 Superdex 75, HiLoad 26/60 Superdex 200, Superdex 200 Increase 10/300 GL) and anionic exchange column (monoQ 10/10).

  28. Amicon Ultra centrifugal filter devices (Millipore) with 10K and 30K molecular weight cutoffs (4.5 mL and 15 mL capacities).

  29. Bradford reagent.

  30. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) using 1X MOPS running buffer (NuPAGE system, Invitrogen). 10% (v/v) polyacrylamide Bis-Tris gels (Invitrogen) (see Note 1).

  31. 4x LDS (Lithium Dodecyl Sulfate) sample dye (adjusted to 200 mM DTT prior to use).

  32. Coomassie stain solution: 40% (v/v) methanol, 10% (v/v) glacial acetic acid, 0.1% (w/v) Coomassie R250.

  33. Destain solution: 10% (v/v) methanol, 10% glacial acetic acid.

  34. Gel imaging apparatus with UV and white light illumination.

2.3. Exo9, Exo13, and Exo14 Core Exosome Reconstitution

  1. Dialysis cassettes (3-12 mL and 0.5 mL volume, 3500 Da cutoff, Thermo-Fisher Slide-A-Lyzer or similar).

  2. Buffer R100: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP-HCl (see Note 2).

  3. Buffer R1000: 20 mM Tris-HCl pH 8.0, 1000 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP-HCl.

  4. Buffer R50: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP-HCl.

3. Protocols

3.1. Cloning

The genetic sequences for Schizosaccharomyces pombe for exosome subunits and cofactors were obtained from the PomBase genome databank [20,21]. In order to achieve high-level expression of these proteins, each of the core subunits and the two exoribonucleases of the RNA exosome (genes rrp41, rrp42, rrp43, rrp45, rrp46, mtr3, rrp4, rrp40, csl4, rrp6, and dis3) were codon optimized for expression in E. coli, synthesized commercially with amenable flanking restriction sites for sub-cloning (Table 1), and acquired within sub-cloning vectors (DNA 2.0/ ATUM, Newark, CA). Additional codon optimized cDNA sequences cti1/rrp47, mpp6, and mtr4 were obtained from IDT DNA and sub-cloned into pGEM sub-cloning vectors.

Table 1:

Summary of expression vectors and purification techniques for S. pombe nuclear exosome components

Protein Vector E. coli Strain Antibiotic Amount of Culture Columns Expected yield
Csl4 pRSF-DUET-His6-Smt3-Csl4 (MCS1) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S75		 210 mg
Rrp4 pRSF-DUET-His6-Smt3-Rrp4 (MCS1) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S75		 100 mg
Rrp40 pRSF-DUET-His6-Smt3-Rrp40 (MCS1) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S75		 160 mg
Rrp41/Rrp45 pRSF-DUET-His6-Smt3-Rrp41 (MCS1)-Rrp45 (MCS2) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S200		 100 mg
Rrp42/Mtr3 pRSF-DUET-His6-Smt3-Rrp42 (MCS1)-Mtr3 (MCS2) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S200		 180 mg
Rrp43/Rrp46 pRSF-His6-Smt3-Rrp43 (MCS1)-Rrp46 (MCS2) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S200		 100 mg
Rrp6/Cti1
pRSF-DUET-His6-Smt3-Rrp6 (MCS1)-Cti1 (MCS2)		 One Shot BL21 Star (DE3) Kan+ 12 L MAC
S200		 60 mg
Dis3 pRSF-DUET-His6-Smt3-Dis3 (MCS1) One Shot BL21 Star (DE3) Kan+ 10 L MAC
S200		 170 mg
Mpp6 pRSF-DUET-His6-Smt3-Mpp6 (MCS1) One Shot BL21 Star (DE3) Kan+ 12 L MAC
S200		 50 mg, <50% pure
Mtr4 pRSF-DUET-His6-Smt3-Mtr4 (MCS1) One Shot BL21 Star (DE3) Kan+ 12 L MAC
S200		 10 mg
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Coexpression strategies for the RNase-PH like subunits (Rrp41, Rrp42, Rrp43, Rrp45, Rrp46, Mtr3) were implemented based on our success in the budding yeast and human systems and facilitated by known x-ray structures for optimal protein production [13,22–25]. This strategy employs coexpression of obligate heterodimeric pairs (i.e. Rrp41/Rrp45, Rrp43/Rrp46, Rrp42/Mtr3, Rrp6/Cti1) to maintain solubility with His6-Smt3 solubility tags located at the N-terminus of the first subunit of the pair. These protein heterodimers can then be purified using affinity and size exclusion chromatography (see Note 3). The remaining subunits were each expressed with N-terminal His6-Smt3 fusions by themselves and also purified by affinity and size exclusion chromatography.

3.1.1. Cloning of S. pombe heterodimers: Rrp41/Rrp45, Rrp43/Rrp46, Rrp42/Mtr3, and Rrp6/Cti1

Genetic sequences encoding for S. pombe rrp41 and rrp45 were inserted into MCS1 and MCS2 of the plasmid pRSF-DUET-His6-Smt3 (Novagen). pRSF-DUET-His6-Smt3 is a modified vector which expresses a His6-Smt3 fusion N-terminal to the protein in the MCS1 (Table 1). Plasmid pRSF-DUET-His6-Smt3-Rrp41(MCS1)/Rrp45(MCS2) encodes two polypeptides, an N-terminal His-tagged Smt3 fusion with Rrp41 and untagged Rrp45. Genetic sequences encoding S. pombe rrp43 and rrp46 were inserted into the MCS1 and MCS2, respectively of the pRSF-DUET-His6-Smt3 vector. Similarly, genetic sequences for S. pombe rrp42 and mtr3 were placed into MCS1 and MCS2 of pRSF-DUET-His6-Smt3, and genetic sequences for S. pombe rrp6 and cti1 (equivalent to S. cerevisiae Rrp47) were also placed into MCS1 and MCS2, respectively, of pRSF-DUET-His6-Smt3. The coexpression strategy is based on using a binding surface between Cti1 and the PMCNT domain of Rrp6 that generates a stable protein complex that can be expressed and purified [13,14,18,26–28]. Quick Change Mutagenesis (Stratagene) was employed to make an exoribonucleolytically inert version of the Rrp6/Cti1 heterodimer by making the residue substitutions Asp243Asn in Rrp6. All plasmids constructed were transformed into the E. coli BL21 One Shot Star (DE3) cell line (Stratagene). Glycerol stocks were prepared from growing up single colonies from these transformations.

3.1.2. Cloning of S. pombe single subunits: Csl4, Rrp4, Rrp40, Dis3, Mtr4, and Mpp6

The S. pombe genetic sequences for S1/KH cap containing components (csl4, rrp4, and rrp40) were each placed into the MCS1 of a pRSF-DUET-His6-Smt3 vector. The genetic sequences for the exoribonuclease dis3, mtr4, and mpp6 were also each placed into the MCS1 of pRSF-DUET-His6-Smt3 construct. Quick Change Mutagenesis (Stratagene) was employed to make an endoribonucleolytically and exoribonucleolytically inert version of the Dis3 protein (by making the residue substitutions Asp166Asn and Asp516Asn) [23]. Each of these plasmids was used to transform E. coli BL21 One Shot Star (DE3) cells (Stratagene). Glycerol stocks were prepared from growing up single colonies from these transformations.

3.2. Protein Expression

3.2.1. Cell Growth and Expression of Exosome subunits

  1. Inoculate one 100 mL flask of LB containing Kanamycin with 50 μl of the respective glycerol stock containing each subunit and grow overnight, shaking at 37°C.

  2. Inoculate 10 shaker flasks of 1 L Super Broth and Kanamycin with 10 mL of the LB overnight culture and grow at 37°C to an OD600 of 1–2. When the OD600 of the culture reaches 1.0-1.5, transfer flask to an ice bath for ~30 minutes and add 20 mL of ethanol to a final concentration of 2%.

  3. Induce protein expression by adding IPTG to a final concentration of 0.25 mM (1 mL of 250 mM stock) and return flasks to an incubator set at 18°C for over night incubation.

  4. Harvest cells by centrifugation at 4000 × g (Beckman JLA-8.1) for 20 minutes at 4°C and discard cleared supernatant. (see Note 4).

  5. Resuspend cell pellets in 50 mM Tris-HCl pH 8.0 and 20% sucrose at a concentration of ~0.5 g cell wet weight per mL.

  6. Distribute resuspended pellets into 50 mL conical tubes, snap-freeze in liquid nitrogen and store at −80°C.

3.3. Protein purification

Below are protocols for preparation for mg quantities of purified exosome components (Table 1). Purification strategies are very similar for all protein preparations: metal affinity purification, cleavage by the Ulp1 protease to cleave off the His6-Smt3 tag, and finally purification by using a size exclusion column and fractionation.

3.3.1. Purification of Exo9 Core Components, exoribonucleases Rrp6/Cti1 and Dis3, cofactor Mpp6, and the helicase Mtr4

  1. Thaw all 50 mL conical tubes of cell suspension from appropriate cell cultures in room temperature water. Add to the cell suspension in Tris-Sucrose buffer: 14 mL 5 M NaCl, 0.8 mL 2.5 M imidazole, 14 μL 14.3 mM BME, 2mL 100 mM PMSF, 2 mL 10% (v/v) IGEPAL ca-630, 200 μL DNAse I (10 mg/mL). This results in the final buffer composition of 350 mM NaCl, 10 mM imidazole, 1 mM BME, 1 mM PMSF, 0.1% (v/v) IGEPAL ca-630, 10 μg/mL DNase I.

  2. On ice/water slurry with rapid stirring, sonicate at 50% output for 3 x 3 minutes with 1 s on 3 s off to disrupt cells.

  3. Pellet cell debris for 45 minutes at 44,000 x g in a JA-20.

  4. Equilibrate 5 mL of Ni-NTA resin with Ni elution buffer.

  5. Transfer resin to one to two 250 mL conical tubes and apply supernatant.

  6. Rotate for 10-30 min.

  7. Transfer the resin to a disposable column and allow the lysate to pass by gravity flow.

  8. Wash resin with ~200 mL Ni wash buffer.

  9. Elute protein with 10 mL Ni elution buffer.

  10. Add 1:1000 molar ratio of Ulp1 and incubate at 4°C overnight for subunit and heterodimeric subunit pairs. Do not add Ulp1 to the preparation of His6-Smt3-Rrp4 sample, as the His6-Smt3 is not removed from Rrp4 until the Exo9 reconstitution step. (see Note 5).

  11. Filter the cleavage reaction/eluate through 0.22 μm and inject over Superdex200 (for Rrp41/Rrp45, Rrp42/Mtr3, Rrp43/Rrp46, Rrp6/Cti1, Dis3, and Mtr4) or Superdex75 (for Csl4, His6-Smt3-Rrp4, Rrp40, and Mpp6) prep grade columns that have been equilibrated with Buffer T350. Collect 5 mL fractions. (see Note 6).

  12. Prepare ‘Fractions’ samples by mixing 30 μL of each fraction with 10 μL of 4x LDS loading dye.

  13. Run 2.5-15 μL of each sample on a 12-well, 10% acrylamide Bis-Tris gel in MOPS-SDS running buffer (180 V limiting, 50 min, room temp).

  14. Transfer the gel to a staining box and stain by gently shaking in Coomassie stain solution for 30 min.

  15. Discard the stain solution in the waste, rinse the gel, and fill with destain solution. Shake for 2-3 hrs and image. A gel of collected fractions for the gel filtration runs for Dis3, Mtr4, Mpp6 and Rrp6/Cti1 is shown in Fig. 1.

  16. Concentrate each of the peak fractions to 5-10 mg/mL in a 15 mL capacity Amicon Ultra 15 (30K MWCO) at 3,000 x g in a hanging bucket rotor. Use an Amicon Ultra 15 10K MWCO for Csl4 and Rrp40. An SDS-PAGE analysis of concentrated samples for the Exo9 core subunits is depicted in Fig. 2A.

  17. Aliquot, flash freeze, and store at −80°C for later use. (see Note 7).

Fig. 1. Purification of nuclear exosome cofactors and exoribonucleases.

Fig. 1

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Coomassie-stained gels of purified and semi-purified core samples. (A) Dis3, (B) Mtr4, (C) Mpp6, and (D) Rrp6/Cti1 samples were each digested with Ulp1 prior to loading onto S200 columns. Traces show absorbance at 280 nm (top panel), and Coomassie-stained gels of the load and fractions near to and including the main peak (bottom panel). Asterisks indicate fractions that were pooled and concentrated.

Fig. 2. Purification of core subunits and reconstitution of S. pombe Exo9.

Fig. 2

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(A) SDS-PAGE analysis of Exo9 subunits used for reconstitution of Exo9 reconstitution. (B and D) A280 trace and Coomassie-stained SDS-PAGE analysis of fractions from Superdex200 gel filtration run of the Exo9 reconstitution. (C and E) A280 trace and Coomassie-stained SDS-PAGE analysis of fractions from monoQ of the Exo9 reconstitution. Each of the purified S. pombe subunits are labeled.

3.4. Reconstitution of S. pombe nuclear exosome and subcomplexes

A three-step strategy is employed to make S. pombe nuclear exosomes (Fig. 3). Using the aforementioned subunits of the core, we reconstitute and purify an Exo9 complex. From this preparation, we further add cofactors, exoribonucleases, and the Mtr4 helicase in two steps to make the Exo13 and Exo14 complexes.

Fig. 3. Reconstitution and purification scheme of S. pombe RNA exosomes.

Fig. 3

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Scheme demonstrates purification of individual subunits to form the Exo9 core. The Exo9 core is then added to the cofactors Mpp6 and Cti1 with exoribonucleases Dis3 and Rrp6 to form an Exo13 sub-complex. The full nuclear Exo14 complex is then formed by the addition of the helicase Mtr4 to the Exo13 sub-complex.

3.4.1. Reconstitution of the Exo9 core

  1. Thaw aliquots of purified Csl4, Rrp40, His6-Smt3-Rrp4, Rrp41/Rrp45, Rrp42/Mtr3, Rrp43/Rrp46 protein samples and place them on ice immediately after the last piece of ice disappears.

  2. Centrifuge proteins at 20,000 x g for 3 min to pellet any material that had precipitated after freeze-thawing.

  3. Measure protein concentration of the supernatants.

  4. Mix the appropriate quantities of core subunits and Ulp1 protease (for cleavage of His6-Smt3-Rrp4 at a mass ratio of ~1/1000) as detailed in Table 2 in T350 Buffer at ~5 g/L in a volume of 5-10 mL. To confirm that all cysteines are fully reduced, also add freshly prepared 10 mM BME to the reconstitution reaction. Do not exceed concentrations above 5 g/L, as higher concentrations will lead to the production of insoluble aggregates during dialysis. Incubate on ice for 30 minutes.

  5. Inject the resulting mixture into a Slide-A-Lyzer dialysis cassette (MWCO 3000) and dialyze over night at 4°C in 2 L of R100 Buffer.

  6. Remove insoluble material by passing the reconstituted mixture through a 0.2 μm filter.

  7. Load sample onto a gel filtration column (Superdex 200 26/60, GE Biosciences) pre-equilibrated with R100 Buffer and collect 5 mL fractions. The complex elutes as a monodisperse peak.

  8. Analyze peak fractions by SDS-PAGE loading 5μl per well (10% Bis-Tris Nu-PAGE gel run in MOPS-SDS Buffer and Coomassie-staining) and pool fractions containing stoichiometric amounts of core subunits.

  9. To further improve the stoichiometry of the complex, apply sample to a Mono Q 10/10 (GE Biosciences) equilibrated with R100. The 9-subunit S. pombe core complex elutes from the Mono Q ion exchange column by applying a linear gradient of NaCl over 20 column volumes from 100 mM NaCl to 600 mM NaCl, eluting at approximately 300 mM NaCl (Fig. 2). Collect 3 mL fractions.

  10. Concentrate each of the peak fractions to 10-15 mg/mL in a 15 mL capacity Amicon Ultra 15 (30K MWCO) at 3,000 x g in a hanging bucket rotor. Expected overall yield for the S. pombe Exo9 core is 10-15 mg.

  11. Aliquot, flash freeze, and store at −80°C for later use.

Table 2:

Sample Exo9 reconstitution

Component Molecular weight (Da) mg/mL μM Multiplier nmol needed μL needed
Csl4 20700 8.7 420 2 300 715
His6-Smt3-Rrp4 48500 6.2 130 2 300 2339
Rrp40 27500 10.1 370 2 300 816
Rrp43/Rrp46 56700 4.0 70 1 150 2117
Rrp42/Mtr3 62200 13.5 220 1.5 225 1036
Rrp41/Rrp45 61000 29.9 490 1 150 306
Ulp1 protease 3 5
Buffer T350 670
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3.4.2. S. pombe Exo13 and Exo14 Reconstitutions

In order to make a nuclear exosome in the S. pombe system, the purified reconstituted core is first added to the ribonucleolytic subunits and cofactors Rrp6/Cti1, Dis3, and Mpp6 to form an Exo13 complex. The Exo13 complex is purified, and then Mtr4 is added to form the Exo14 complex and purified.

  1. Thaw aliquots of purified Rrp6/Cti1, Dis3, Mpp6, and reconstituted/purified Exo9 and place them on ice immediately after the last piece of ice disappears.

  2. Centrifuge proteins at 20,000 x g for 3 min to pellet any material that had precipitated after freeze-thawing.

  3. Measure protein concentration of the supernatants.

  4. Mix the appropriate quantities of Exo9, Rrp6/Cti1, Dis3, and Mpp6 as detailed in Table 3 in T350 Buffer at ~10 g/L in a volume of 250 to 500 μl for small scale reconstitutions or up to 5 mL for large scale reconstitutions. Incubate on ice for 30 minutes.

  5. Inject the resulting mixture into a Slide-A-Lyzer dialysis cassette (MWCO 3000) and dialyze over night at 4°C in 2 L of R100 Buffer.

  6. Remove insoluble material by passing the reconstituted mixture through a 0.2 μm filter.

  7. Load sample onto a gel filtration column (Superdex 200 Increase 10/300 GL for small scale or Superdex 200 26/60 for large scale, GE Biosciences) pre-equilibrated with R100 Buffer and collect 5 mL fractions. The complex elutes as a monodisperse peak (Fig. 4A).

  8. Analyze peak fractions by SDS-PAGE loading 5 μl per well (10% Bis-Tris Nu-PAGE gel run in MOPS-SDS Buffer and Coomassie-stained; see Subheading 3.3.1 steps 12–15) and pool fractions containing stoichiometric amounts of core subunits.

  9. Concentrate each of the peak fractions to 10-15 mg/mL in a 15 mL capacity Amicon Ultra 15 (30K MWCO) at 3,000 x g in a hanging bucket rotor. Expected overall yield for the S. pombe Exo13 core is ~3 mg. Immediately proceed to Subheading 3.4.2 step 10 for reconstitution of Exo14. Otherwise, aliquot Exo13, flash freeze, and store at −80°C.

  10. Add 1.5 molar excess of Mtr4 to freshly prepared Exo13 in T350 Buffer at ~10 g/L in a volume of ~0.5 mL. Precise amounts of material are depicted in Table 4. Incubate on ice for 30 minutes.

  11. Inject the resulting mixture into a Slide-A-Lyzer dialysis cassette (MWCO 3000) and dialyze for 6 hours at 4°C in 2 L of R100 Buffer and then over night at 4°C in 2 L of R50 Buffer.

  12. Remove insoluble material by passing the reconstituted mixture through a 0.2 μm filter (see Note 8).

  13. Load sample onto a gel filtration column (Superdex 200 Increase 10/300 GL) pre-equilibrated with R50 Buffer and collect 0.5 mL fractions. The complex elutes as a monodisperse peak (Fig. 4B).

  14. Analyze peak fractions by SDS-PAGE loading 5 μl per well (10% Bis-Tris Nu-PAGE gel run in MOPS-SDS Buffer and Coomassie-stained; see Subheading 3.3.1 steps 12–15) and pool fractions containing stoichiometric amounts of core subunits (see Note 9).

  15. Concentrate each of the peak fractions to 10-15 mg/mL in a 4 mL capacity Amicon Ultra 4 (30K MWCO) at 3,000 x g in a hanging bucket rotor. A typical expected overall yield for the S. pombe Exo14 complex is 0.5-1 mg.

  16. Aliquot Exo14, flash freeze, and store at −80°C.

Table 3:

Sample Exo13 reconstitution

Component Molecular weight (Da) mg/mL μM Multiplier nmol needed μL needed
Exo9 266000 16.6 62 1 4 64.0
Dis3 110000 24.6 220 1.2 4.8 21.6
Rrp6/Cti1 105000 31.5 300 1.5 6 20.0
Mpp6 21500 13.1 610 3 12 19.8
Buffer T350 124
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Fig. 4. Reconstitution and purification of S. pombe Exo13 (A and C) and Exo14 (B and D).

Fig. 4

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A280 traces (A and B) and Coomassie-stained SDS-PAGE analysis of fractions (C and D) from each gel filtration run from the two reconstitutions. All S. pombe subunits of Exo14 are labeled.

Table 4:

Sample Exo14 reconstitution

Component Molecular weight (Da) mg/mL μM Multiplier nmol needed μL needed
Exo13 503000 11.8 23 1 1.9 81.0
Mtr4 126000 25 20 1.5 2.85 14.4
Buffer T350 45
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Acknowledgements

We thank Lima Lab members for advice during the course of this work, and Fangyu Liu for her contributions to reconstituting S. pombe exosomes. This work was supported in part by GM079196 and GM118080 (NIH/NIGMS, C.D.L) and P30CA008748 (NIH/National Cancer Institute). The content is the authors’ responsibility and does not represent the official views of the NIH. C.D.L is a Howard Hughes Medical Institute Investigator.

4. Notes

1.

Many different gel percentages were used. We found the combination of 10% acrylamide with 1X SDS-MOPS provided the best separation for all 14 components of the entire nuclear exosome reconstitution.

2.

As a more affordable option, beta-mercaptoethanol can also be used in lieu of TCEP-HCl in reconstitution buffers. It was used at a concentration of 5 mM for all reconstitution buffers (R100, R1000, and R50). However, beta-mercaptoethanol should be added fresh to these buffers right before performing reconstitutions and purifications.

3.

The order of the heterodimeric pairs within MCS1 and MCS2 for the RNase-PH like subunits is crucial for purification of both subunits. For instance, we tried cloning and expressing heterodimeric pairs in the opposite MCS positions as shown in Table 1 (i.e. Rrp45 (MCS1)/ Rrp41 (MCS2), Rrp46 (MCS1) / Rrp43 (MCS2), and Mtr3 (MCS1) / Rrp42 (MCS2)), and we were unable to express either component or only could express/purify the subunit that was tagged.

4.

Add antifoam during the spin-down/harvest step (i.e. post induction). We have found the addition of anti-foam during induction has deleteriously impacted the expression levels of many of the S. pombe exosome subunits. However, we still add antifoam at a concentration of 1/10,000 (v/v) post-induction in order to retain sample that would otherwise be lost as foam.

5.

Rrp4 has low solubility without the His6-Smt3. It can be purified without the tag, but the yield is greatly reduced.

6.

The Rrp42/Mtr3 heterodimer produces three bands. One band that corresponds to Rrp42 and two bands that correspond to Mtr3 (Fig. 2). One band for Mtr3 can be detected at ~55 kDa (Mtr3-1) and another at ~28 kDa (Mtr3-2). The expected molecular weight for Mtr3 is 28,400 Da. The strange pattern is also detected when using other acrylamide percentages and buffer types. However, the apparent higher molecular species is not present when reconstituted into Exo13 and Exo14 complexes. In these complexes, Mtr3 runs near its expected size.

7.

Catalytically inert versions of Dis3 and Rrp6/Cti1 use an identical protocol to the one described here for wild-type proteins.

8.

For reconstitutions that are less than 200 μl, remove insoluble material by spinning at 20,000 x g for 1 min. A significant fraction of your material would be lost at this volume by passing the sample through a filter.

9.

Be very careful when pooling fractions. The reconstituted species runs very close to a small but significant void peak (Fig. 4B). If fractions from the void peak are mixed in with the desired monodisperse peak, the entire sample will aggregate during concentration and decrease the quality and yield of the Exo14 reconstitution.

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