Olivier Pellegrini

Laetitia Gilet

Aude Trinquier

Anastasia Tolcan

Delphine Allouche

Sylvain Durand

Frédérique Braun

Ciarán Condon

Assay of Bacillus subtilis ribonuclease activity in vitro

Keywords: Ribonuclease, protein purification, RNA substrates, in vitro transcription, radioactive labelling

Ribonucleases can cleave RNAs internally in endoribonucleolytic mode or remove one nucleotide at a time from either the 5' or 3' end through exoribonuclease action. To show direct implication of an RNase in a specific pathway of RNA maturation or decay requires the setting up of in vitro assays with purified enzymes and substrates. This chapter complements the previous chapter X on assays of ribonuclease action in vivo by providing detailed protocols for the assay of B. subtilis RNases with prepared substrates in vitro.

Introduction

The Gram-positive model organism Bacillus subtilis has >20 different ribonucleases (RNases) involved in mRNA and stable RNA maturation and decay [1]. Remarkably, fewer than half of these RNases overlap with the well-studied E. coli model. In B. subtilis, the main endoribonucleases involved in mRNA decay are RNase Y, which cleaves in single stranded AU-rich regions, and RNase III, which cleaves dsRNA. The main 5'-exoribonuclease is RNase J1, which functions in a stoichiometric complex with its paralog RNase J2, while the main 3'-exoribonucleases are PNPase, RNase PH, RNase R and YhaM. rRNA processing involves RNase III, RNase J1, YqfG, Mini-III and RNase M5 in B. subtilis, while tRNA processing requires RNase P and either RNase Z or the four 3'-exoribonucleases, depending nominally on whether the tRNA has an encoded CCA.

RNases are routinely assayed in vitro to definitively demonstrate that the effect of an enzyme observed in vivo is direct rather than indirect (see chapter X). In vitro assays are also used to establish an enzyme's biochemical (Km, Kd) or kinetic parameters (Kcat). We generally use purified His-tagged enzymes for these assays. For new uncharacterized enzymes, we typically start with standard buffer at neutral pH, 100 mM NaCl, 10 mM Mg 2+ as the most likely metal ion and a reducing agent (e.g. dithiothreitol [DTT]/mercaptoethanol) and then optimize each of these buffer components in turn. Although assays can be performed on generic RNA targets, it is preferable that the true substrates be used. Our standard operating procedure is to synthesize specific RNA substrates by transcribing PCR products containing an integrated phage T7 promoter. These can be labelled with -32 P-UTP directly or can be synthesized as unlabelled substrates and labelled later at their 5' or 3' extremities using -32 P-ATP or -32 P-pCp. For very short RNA substrates (< 50 nucleotides [nts]), we order chemically synthesized RNA oligonucleotides and label these at their 5' or 3' extremities. Strategies also exist for non-radioactive labelling, for example using fluorescent or digoxygenin labelled probes, although we do not use these very often, because they are less sensitive than radioactively labelled substrates In this companion chapter to chapter X, we describe how we typically assay RNase activities in vitro. We describe in depth how we prepare His-tagged RNases, their RNA substrates and finally how we set up the in vitro assays themselves. We provide detailed notes on all our lab secrets to succeed with these procedures.

Materials

23. 1 L Dialysis buffer: 20 mM Tris-HCl pH 7.8, 0.3 M NaCl, 10% v/v glycerol, 0.5 mM dithiothreitol (DTT). Filter at 0.22 µm. Store at 4°C. [START_REF]RNA and no incubation (C0)[END_REF]. Kit for measuring protein concentration (e.g. Protein assay kit II from Biorad).

Dialysis chambers or tubing.

26. Centrifugal filter concentration units (e.g. Amicon units from Millipore) with molecular weight cutoff adapted to the RNase of interest.

Protein gels (SDS-PAGE)

1. Gel electrophoresis apparatus with gel casting system (e.g. Biorad Mini Protean 2) and power supply. 12. 4X Stacking buffer: 0.5 M Tris-HCl pH 6.8, 0.4 % (w/v) SDS.

13. 10% acrylamide solution: for 10 mL, mix 2.5 mL of 4X resolution buffer with 2.6 mL of 40% acrylamide:bis-acrylamide (37.5:1) solution. Complete to 10 mL with deionized H 2 O. Vortex the solution.

14. 5% acrylamide solution: for 5 mL, mix 1.25 mL of 4X stacking buffer with 0.625 mL of 40% acrylamide:bis-acrylamide (37.5:1) solution. Complete to 5 mL with deionized H 2 O. Vortex the solution.

15. 10% (w/v) ammonium persulfate (APS). Make 1 mL aliquots and store at -20 °C. 16. N,N,N',N'-Tetramethylethylenediamine (TEMED). 17. 5X Gel loading buffer: 0.312 M Tris-HCl pH 6.8, 10 % (w/v) SDS, 0.5 M DTT, 50% (v/v) glycerol, 0.04 % (w/v) bromophenol blue. 18. Plastic containers for gel staining. 18. Liquid nitrogen and long insulated gloves for handling it safely.

RNase assays in vitro

1. Sterile RNase-free microcentrifuge tubes.

2. Gel electrophoresis system with 40 x 34 cm glass plates, 0.4 mm spacers and 32-well gel comb for polyacrylamide gel electrophoresis.

3. Power supply.

4. Screens and protective clothing to work with 32 P. 5. PhosphorImager with 32 P-phosphorimaging screen and cassette (GE Healthcare).

6. Mini-sub-cell system (BioRad) with gel tray, comb, tank, and gel mold, or equivalent system for the preparation and migration of agarose gels (15 x 7 cm).

Methods

Cultures for overproduction of His-tagged proteins

Affinity tags are highly efficient tools for purifying proteins. One of the most used is the Histag, which provides good yields of tagged protein from inexpensive, high capacity resins. We commonly use Nickel or Cobalt Talon (Co-Talon) affinity metal resins, depending on the protein solubility with respect to pH (see Note 1). Both can be employed in native or denaturing conditions (see Notes 5, 6).

1. Inoculate 20 mL of growth medium with the E. coli RNase overproducing strain.

2. Incubate at 37°C overnight with shaking.

3. Use 4 mL of overnight culture to inoculate 400 mL of fresh growth medium and grow at 37°C with shaking to an optical density at 600nm (OD 600 ) of 0.4-0.6 (see Notes 7, 8). 4. To induce protein expression, add 200 µL of 1M IPTG (0.5 mM, final concentration) and incubate at 37°C under shaking for 3-4 h. 5. Pellet cells in 250 mL centrifuge bottles for 20 min at 9,000 g at 4°C.

6. Pour off all but about 20 mL of supernatant.

7. Carefully dislodge the pellet with a spatula and transfer pellet and remaining supernatant to a 50 mL conical tube.

8. Centrifuge for 15 min at 5,000 g at 4°C. 9. Remove supernatant and store the pellet frozen at -20°C until further use.

Purification of His-tagged proteins under native conditions

Wear gloves to avoid contamination of protein samples.

1. Resuspend the frozen cells in 10 mL of Binding buffer at 4°C. 2. Add 10 µL 10 mg/mL DNase I and an EDTA-free anti-protease tablet.

3. Before making cell lysate, install the His-Trap FF on the FPLC system and start equilibration of column with Binding buffer at 1 mL/min (see Note 9). 4. Pass the suspension (from step 2) twice through a French press (20,000 psi).

5. Centrifuge the lysate for 30 min at 15,000 g and keep supernatant. 8. Wash the column with approximately 10 mL Binding buffer. Collect this fraction in a 15 mL tube for analysis. 9. Wash with 15 mL Wash buffer. Collect this fraction in a 15 mL tube for analysis (see Note 2) 10. Apply 10 mL Elution buffer to the column and collect 1 mL fractions in 1.5 mL microtubes (see Notes 2, 10).

11. Confirm the protein peak by measuring protein concentration using a dedicated kit. Keep small aliquots (e.g. 50 L) of these fractions for analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Section 3.3).

12. Pool the remainder of the peak fractions and dialyze immediately overnight against Dialysis buffer (see Note 11). 13. Concentrate samples if necessary with filter concentration units, following manufacturer's instructions.

14. Analyze different fractions of the purification scheme by SDS-PAGE.

15. Measure protein concentration with a dedicated kit, aliquot and store at -80°C.

Protein gels (SDS-PAGE)

SDS-PAGE is a technique designed to dissociate proteins into their individual polypeptide subunits by denaturation in the presence of detergent (SDS). Since the charge contribution is negligible compared to that of the negatively charged SDS, it is also possible to determine the molecular weight of the polypeptides by comparing their migration position to that of a molecular weight standard.

1. Clean the glass plates with a few milliliters of 95% ethanol and assemble the 1 mm wide, (10 x 8) cm gel cassette, following manufacturer's instructions.

2. Mix 10 mL of the 10% polyacrylamide gel solution with 50 µL 10% APS and 10 µL TEMED and pour this "resolution gel" mixture into the gel cassette. 19. Store gel in Conservation solution.

Preparation of substrates for RNase assays

All materials and solutions must be RNase-free and gloves should be worn for the entirety of all procedures.

T7 in vitro transcription

We generally use PCR templates with an integrated T7 RNA polymerase promoter as a DNA template for in vitro transcription reactions. The minimal sequence of T7 RNA polymerase promoter is 5'-ATTAATACGACTCACTATAG-3' for transcripts that start with a G and 5' CAGTAATACGACTCACTATTA 3' for transcripts that start with an A. To increase the yield of in vitro transcription, add one or two extra guanines to the 3' end of the minimal promoter sequence, e.g. GGG or AGG, but it is important to note that this addition will be incorporated into the 5' end of the transcript. Linearized plasmid DNA that contains a T7 RNA polymerase promoter site can also be used as a template for in vitro transcription.

Production of unlabelled T7 in vitro transcripts with various 5' ends.

We have adapted the MEGA(Short)Script kit protocol to obtain different 5' ends (5' triphosphate, 5'-monophosphate, 5'-hydroxyl) on RNA transcripts. For short transcripts (<150 nts), we use the MEGAShortScript kit; for those that are longer, we use the MEGAScript kit. 5. Determine RNA concentration from UV absorbance at 260 nm using an extinction coefficient of 0.025 (μg/ml) -1 cm -1 .

Production of -32 P-UTP labelled T7 in vitro transcripts

To produce uniformly labelled transcripts, we add a lower concentration of cold UTP (60µM) while keeping the rest of rNTPs at 2.5mM (see Note 16). Since the yields required are typically not that high, we use individually purchased enzymes and nucleotides rather than the more expensive kits for this purpose. All radioactive materials should be handled by properly trained scientists, following safety rules and local regulations.

1. In a sterile, RNase-free 1.5 mL microtube, mix 0.1 -0.2 µg linear DNA template, 2.5 µL 100 mM DTT, 5 µL of rNTP label mix (2.5 mM each ATP, CTP GTP and 60 µM UTP), Synthetized RNA In a sterile, RNase-free 1.5 mL microtube, mix 10 to 40 pmol dephosphorylated RNA, 2 µL 10X T4 PNK kinase buffer, 3 µL γ-32 P-ATP, 9 µL RNase-free water, 2 µL T4 PNK kinase.

12. Incubate 30 min at 37°C in a water bath.

13. Clean up unincorporated nucleotides on a G50 spin column (following manufacturer's recommendations) or gel purify 32 P-labeled RNA (Section 3.3.6).

3'-end labelling of RNA substrates

1. In a sterile, RNase-free 1.5 mL microtube, mix 10 µL 10 µM RNA (from Section 3.3.2, step 5), 2.5 µL DMSO, 1.25 µL 10 mM ATP, 2.5 µL 10x T4 RNA ligase buffer, 1.25 µL 40U/µL RNasin, 3 µL -32 P-pCp, 2.5 µL RNase-free sterile water, 2 µL T4 RNA ligase.

2. Incubate for 4h at 16°C or overnight at 4°C.

3. Incubate the mixture for 15 min at 65°C in a dry temperature block to inactivate the T4 RNA ligase.

14. Clean up unincorporated nucleotides on a G50 spin column (following manufacturer's recommendations) or gel purify 32 P-labeled RNA (Section 3.3.6).

Gel purification of RNA

Non-homogenous PCR templates, abortive T7 RNA polymerase transcription products or incomplete chemical synthesis of RNA can lead to the appearance of labelled contaminating species that are shorter than the desired transcript. In this case, full-length (FL) labelled RNA can be purified from a polyacrylamide gel (from 5 to 20% depending on the size of the RNA).

1. To pour a vertical polyacrylamide gel (0.4 x 400 x 340 mm), treat the smaller glass plate with a few milliliters of silanization solution and spread evenly with a paper towel under a fume hood.

2. Mix 70 mL of the polyacrylamide gel solution with 700 µL 10% APS and 70 µL TEMED. Pour gel in a preassembled 0.4 x 400 x 340 mm gel cassette and insert a 32well gel comb immediately, without introducing air bubbles. Allow the gel to polymerize for 1 h at room temperature.

3. Mount gel on the electrophoresis apparatus and fill reservoirs with 0.5X TBE buffer.

4. Rinse the gel wells with a Pasteur pipette filled with 0.5X TBE to remove urea and polyacrylamide fragments.

5. Add the same volume of 2X RNA dye to the 32 P labelled RNA samples and load all the sample, occupying more than one lane if necessary.

6. Run the gel at 60W to separate the full-length transcript from contaminating truncated species.

7. Remove one of the glass plates and cover the gel in Saran Wrap TM to prevent it from drying.

8. Expose the gel to an X-ray film in a dark room (1-10 minutes depending on efficiency of radioactive labelling), taking care to align the edge of the film with the top and one of the side edges of the glass plate. Fold down one corner of the film for orientation.

9. Develop the film.

10. Place the developed film with the visible radioactive band under the glass plate, in the correct orientation, aligning the film to the top and side edges as before, to precisely locate the portion of the gel to cut out.

11. Cut the band out (through the Saran Wrap TM ) as tightly as possible with a clean new scalpel, peel off the Saran wrap and place the radioactive gel slice in 400 µL of 0.3 M sodium acetate in a 1.5 mL microtube. This can all be done with the same scalpel and clean forceps.

12. Elute on a rotating wheel for 4h in the cold room.

13. To precipitate RNA, add 1 mL 95% ethanol, 1 µL 20 mg/ml glycogen and incubate for at least 1 hr at -20°C.

14. Centrifuge for 30 min at 20 000 g at 4°C. Discard supernatant.

15. Add 200 uL 70% ethanol (-20°C) to pellet 16. Centrifuge for 30 min at 20 000 g at 4°C. Remove supernatant carefully. 17. Add 200 uL 95% ethanol (-20°C) to pellet 18. Centrifuge for 30 min at 20 000 g at 4°C. Remove supernatant carefully.

19. Resuspend RNA pellet in 20-50 µL nuclease free water.

Preparation of ribosome precursors

In addition to assays using in vitro transcribed RNA substrates, RNase activity can be assessed on more complex substrates isolated in vivo. This is particularly useful for the study of RNases involved in ribosomal RNA (rRNA) maturation that recognize components of the ribosome. For example, RNase M5 cleavage is dependent on prior binding of L18 to the pre-12. Pool fractions of interest according to the absorbance profile at OD 260nm (see Notes 20,

21).

13. To concentrate precursor particles, centrifuge at 264,500 g in a TLA 100.3 rotor in polycarbonate centrifuge tubes.

14. The ribosomal pellet obtained after ultracentrifugation is transparent and very sticky, make sure not to touch it with the pipette tip while removing all the supernatant.

15. Resuspend pellet in 50 to 100 µL 1X Buffer A by vortexing gently in cold room for 20 min (or until all the ribosomal pellet is resuspended).

16. Freeze aliquots in liquid nitrogen and store at -80°C.

RNase assays in vitro

When doing in vitro assays, we typically first check the effect of different protein concentrations on the degradation/maturation of the substrate in a dose response assay. Once we have identified an appropriate enzyme concentration that functions well, we often switch to kinetic reactions to identify subtle differences between wild-type and mutant variants, for example. As substrates, we use radiolabeled RNAs, cold transcripts or more complex substrates such as ribosome precursors (see previous section). While the enzyme reactions can be followed directly on gels with labelled substrates, with unlabelled substrates, the assays are followed by Northern blots (see chapter X, section 3.3). Here, we detail a typical protocol to follow the release of a 32 P-labelled 5'-monophosphate by the B. subtilis 5'exoribonuclease RNase J1 from a 20 nt RNA, as an example [5]. For other enzymes, the optimal buffer, substrate and general reaction conditions might be different.

Dose-response assay

1. To find the optimal RNase J1 concentration for an in vitro assay, first prepare a dilution of the enzyme at 1 mg/mL using enzyme Dilution buffer in 1.5 mL microtubes and then make 10 -1 , 10 -2 and 10 -3 dilutions of this preparation for assays (see Notes 22, 23). Keep the tubes on ice while preparing the reaction mixtures. Each 10 µL reaction will contain 1 µL of diluted RNase J1 , 0.5 µL 5'-labelled RNA, 2 µL 5X RNase J1 buffer, 6.5 µL nuclease-free water.

2. In a 1.5 mL RNase free microcentrifuge tube, prepare a reaction mix for n+1 samples (without the protein). For instance, mix 3.5 µL 5'-labelled RNA, 14 µL 5X RNase J1 buffer and 45.5 µL nuclease-free water for 6 samples (e.g. four reaction samples and two negative control samples). 2. If protein solubility permits, a more stringent wash and elution protocol involving a pH gradient instead of imidazole can be used. After binding, an alternative wash buffer (50 mM NaH 2 PO 4 pH 6.0, 0.3 M NaCl) is applied to the column, and the protein is eluted with 10 mL low pH elution buffer (50 mM sodium acetate pH 4.0, 0.3 M NaCl, 250 mM Imidazole).

3. Add -Mercaptoethanol to Buffer A just before use.

1X Buffer

A is a typical ribosome storage buffer. The protocol given here, using 1X

Buffer A with 3 mM MgCl 2 , is designed to dissociate the small (30S) and large (50S) ribosomal subunits. 1X Buffer A with high magnesium concentration (10 mM MgCl 2 ) is required to maintain assembled 70S ribosomes.Denaturing conditions can be used for proteins that form inclusion bodies or for soluble proteins whose His-tag is not accessible for interaction with the Ni-NTA resin under native conditions. In the first case, most of the overproduced protein is found in the pellet of cellular debris after the first centrifugation step following cell lysis. In the second case, the overproduced protein remains in the supernatant after the first centrifugation step but is found primarily in the flow-through (FT) and wash fractions after application to the Ni-NTA agarose column.

Proteins that form inclusion bodies are solubilized from the pellet of cell debris in 5 ml binding buffer at room temperature. Cell debris is recentrifuged at 15,000 g for 20 min at room temperature. For soluble proteins whose His-tag is not available, the frozen cells are resuspended in 10 mL of binding buffer at room temperature. The suspension is centrifuged for 30 min at 15,000 g at 4°C. Steps 6-11 of the native purification protocol (section 3.2) are then applied. 6. Instead of the 2XYT medium, the use of tryptone phosphate medium has been described

to prevent the inclusion body formation: 2% bactotryptone, 0.2% Na 2 HPO 4 , 0.1% KH 2 PO 4 , 0.8% NaCl, 1.5 % yeast extract, 20% glucose [6].

7. To avoid the formation of inclusion bodies, it is also possible to induce the culture at a temperature of 16°C for 16 h.

8. In the absence of an FPLC system, home-made columns can be packed using commercial Ni-NTA (Qiagen) or Co-Talon resins (Clontech) in 1 mL syringes, plugged with a circular piece of Whatman 3MM paper. Flow-rate is controlled at 10 mL/h by a peristaltic pump and fractions are collected using a basic model fraction collector (e.g.

Frac-200, GE Healthcare). 9. At higher concentrations, certain proteins tend to aggregate and precipitate. In this case, it is possible to dilute the protein immediately after elution by adding buffer to the fraction collection tubes.

10. Some RNases (e.g. RNase Z) are rapidly inactivated in the presence of imidazole. We therefore do not wait for the result of the SDS-PAGE analysis before dialyzing.

11. It is possible to knot a piece of tissue paper and place in reservoir to accelerate the destaining process by absorption of the remaining dye.

2 .

 2 Dry temperature block. 3. Orbital shaker. 4. Latex gloves or equivalent. acrylamide:bis-acrylamide (37.5:1) solution. 10. 1X Running buffer: 0.025 M Tris, 0.192 M glycine, 0.1 % (w/v) sodium dodecyl sulfate (SDS) in deionized H 2 O. 11. 4X Resolution buffer: 1.5 M Tris-HCl pH 8.8, 0.4 % (w/v) SDS.

18 .

 18 5X RNase J1 buffer: 100mM Tris-HCl pH 8, 40 mM MgCl 2 , 500mM NH 4 Cl, 0.5mM DTT. 19. RNA substrate: 20 nts synthetic oligoribonucleotide, 5' or 3' radiolabeled (see Section 3.3.4) and gel purified (see Section 3.3.5). 20. Nuclease-free water 21. 20% acrylamide solution: dissolve 420 g urea in 500 mL 40 % acrylamide:bisacrylamide (19:1) solution and 100 mL 10X TBE buffer (from commercial source). Complete to 1L with RNase-free water. Filter solution on a 0.22 µm filter and keep at 4°C in a dark bottle. 22. Solutions 18 -22 from Section 2.3.1. 23. Whatman 3 MM paper cut to the size of the gel. 24. Vacuum gel dryer and vacuum pump.

6 .

 6 Add 1/500 volume of 0.5 M imidazole-HCl (pH 7.8) to the supernatant to give a final concentration of 1 mM. 7. Apply the resulting mix to the column at the same flow rate as during equilibration.

3 . 7 .

 37 Layer a few drops of isobutanol to the unpolymerized gel to ensure the top surface of the gel is flat.4. Polymerize the gel for 1 h at room temperature.5. Wash the gel surface thoroughly with deionized water to remove isobutanol. 6. Mix 5 mL of the 5% polyacrylamide gel solution with 25 µL 10% APS and 10 µL TEMED. Pour this "stacking gel" mixture on top of the resolution gel and insert the comb immediately, without introducing air bubbles.8. Polymerize the gel for 1 h at room temperature. 9. Mount gel on apparatus and fill reservoirs with 1X Running buffer. 10. Mix 4 volumes of protein sample with 1 volume of 5X gel loading buffer. The final volume should not exceed 25 µL. 11. Heat the samples for 3 min at 90°C in a dry temperature block and load them in the gel wells. 12. Run the gel at room temperature at 20 mA. Stop the migration when the bromophenol blue dye reaches the bottom of the gel. 13. Separate the gel plates with the help of a spatula and place the gel in Staining solution in a plastic container. 14. Shake slowly on orbital shaker for 30 min. 15. Remove Staining solution and rinse the gel with deionized water. 16. Add 20 mL of the fast Destaining solution and shake slowly for 1 h. 17. Remove fast Destaining solution and rinse the gel with deionized water. 18. Add 20 mL of the slow Destaining solution and shake slowly for a few hours until the desired color is obtained (see Note 12).

3 .

 3 Add 2 l of TURBO DNase (from the MEGA(Short)Script kit), then incubate for 15 minutes at 37°C to degrade the DNA template.

4 .

 4 Clean up unincorporated nucleotides on a G50 spin column (following manufacturer's recommendations) or gel purify the RNA transcript (Section 3.3.6).

6 . 7 . 8 . 9 . 1 .

 67891 Add enough RNase J1 enzyme to the step 3 mixture for n-1 aliquots and mix. In the example given, this would correspond to 3 µL (0.5 µL per time point) at the appropriate dilution determined from the dose response curve in Section 3.4.1. Incubate the C30 control and the reaction mixture (from step 6) at 37°C. Remove 5 µL sample aliquots at 2, 5, 10, 20 and 30 min from the reaction mixture and add each aliquot to a tube from step 1. Store aliquots on ice. After 30 min of incubation, also add 5 µL 2X RNA loading dye to the C30 control and put on ice. Incubate samples for 5 min at 90°C to denature them. Then load each 10 µL sample into a well of the 20% polyacrylamide gel. Run the gel at 60W in 1X TBE.[START_REF]Air dry the pellet and resuspend it in 10 uL RNase-free water. Determine RNA concentration from UV absorbance at 260 nm using an extinction coefficient of 0[END_REF]. Process and dry gel as described in Section 3.4.1, steps 9-13 (see Notes 28, 29). A typical kinetic assay profile is depicted in Figure2. [Figure2near here] Cobalt-Talon columns have been used to purify His-tagged proteins to a higher degree of purity than HisTrap columns in some cases. In this case the following buffers are used: Binding buffer: 50 mM sodium phosphate pH 7.0, 0.3 M NaCl, 0.1 % (v/v) Triton X-100, 10 % (v/v) glycerol; Wash buffer: 50 mM sodium phosphate pH 7.0, 0.3 M NaCl, 20 mM imidazole ; Elution buffer: 50 mM sodium phosphate pH 7.0, 0.3 M NaCl, 150 mM imidazole The protein can also be eluted from Co-Talon columns with a low pH elution buffer containing 50 mM sodium acetate pH 5.0, 0.3 M NaCl, and 150 mM imidazole.

5 .

 5 Buffers for purification under denaturing conditions are as follows: Binding buffer: 100 mM sodium phosphate, 10 mM Tris-HCl pH 7.8, 6 M guanidine. Wash buffer: 100 mM sodium phosphate, 10 mM Tris-HCl pH 6.3, 6 M guanidine. Elution buffer: 100 mM sodium phosphate, 10 mM Tris-HCl pH 4.5, 6 M guanidine.

Figure 1 .

 1 Figure1. Schematic representation of an RNase J1 5' exoribonuclease dose response assay measuring release of 32 P-NMP from the 5' end of a radiolabeled 20 nt substrate in the presence of a 10-fold serial dilution of the enzyme. The higher concentration, the stronger the activity observed. The activity is 100 times weaker in a catalytic mutant.

Figure 2 .

 2 Figure 2. Schematic representation of an RNase J1 5' exoribonuclease kinetic assay. 32 P-NMP is released after 2 min and reaches a plateau at 10 min.

  1. Prepare transcription mixture according to the following table in 1.5 mL microtubes (see

	Notes 13, 14).

2. Incubate mixture for at least 3h at 37°C in a water bath (see Note 15).

Acknowledgments

This work was supported by funds from the CNRS (UMR 8261), Université de Paris, the Agence Nationale de la Recherche (ARNr-QC; BaRR) and the Labex (Dynamo) program.

then peel off the gel attached to the Saran Wrap TM and then apply Whatman 3MM paper to the gel for drying.

28. If the substrate studied is not radiolabeled, once the migration is over, transfer the gel to a nitrocellulose membrane Amersham Hybond TM -N + . Probe the RNA with a specific radiolabeled probe, standard Northern Blot protocol, see Chapter X, Section 2.3 for details.


Laetitia Gilet

Olivier Pellegrini

Anastasia Tolcan

Delphine Allouche

Sylvain Durand

Frederique Braun

Ciarán Condon

Aude Trinquier

Frédérique Braun

Assay of Bacillus subtilis ribonuclease activity in vivo

Keywords: Ribonuclease, bacterial cell cultures, rifampicin kinetics, Northern blot, capillary transfer, electrotransfer, hybridization

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ribonucleases (RNases) play key roles in remodelling RNA, by cutting it internally or progressively shortening it from either end (for review see [1]). This can have major consequences for gene expression. Cleaving an mRNA by an endoribonuclease within a coding sequence, for example, completely inactivates the RNA, rendering it useless for translation and typically sending the resulting RNA species on a pathway to full degradation.

On the other hand, cleaving an mRNA within its 5' or 3 untranslated regions (UTRs), or in the intergenic regions between cistrons, can have either stabilising or destabilising effects, depending on the proximity of these cleavages to potential protective secondary structures, with a parallel effect on gene expression. Indeed, secondary structures at the extreme 5' or 3' end of RNAs typically protect them from degradation by exoribonucleases (and some 5'-enddependent ribonucleases).

RNase cleavage of stable RNAs can also have a major impact on both their functionality and stability. The trimming of extraneous RNA from ribosomal RNA (rRNA) or transfer RNA (tRNA) precursors is a critical step in their becoming functional. Removal of 3' extensions from tRNAs precursors, for example, allows the universal CCA motif to become available for aminoacylation. Similarly, removal of the 3' extension of 16S rRNA improves the availability of the anti-Shine-Dalgarno (SD) sequence to bind to the SD sequence of the ribosome binding site of bacterial mRNAs. Failure to correctly process these RNAs, not only renders them less or non-functional, but also marks them for degradation by 5' and 3' exoribonucleases that use the extensions as landing pads to begin the decay process [2]. This is a recurring theme in quality control degradation pathways for non-functional stable RNAs.

Lastly, processing of small regulatory RNAs (sRNAs) can, like mRNAs and stable RNAs, influence both their functionality and stability. We have recently shown, for example, that the bacterial RoxS sRNA interacts better with its target mRNA sucCD when cleaved by an endoribonuclease at position 20 [3]. This processing event is not necessary for interactions with other targets such as the ppnKB mRNA. Intriguingly, cleavage at this position is also the first step in the degradation of this sRNA. Thus, it appears that this degradation intermediate can be at least temporarily side-tracked from its ultimate fate to perform a specific regulatory function.

Understanding the roles of RNases in the maturation and degradation of mRNAs, stable RNAs or sRNAs requires measurement of half-lives or observation of differences in the sizes of RNA species in wild-type (wt) vs. mutant strains by Northern blot. In this chapter, we describe our current laboratory protocols for measuring mRNA half-lives in the Gram-positive model B. subtilis. In the accompanying chapter Y, we describe how to assay RNase activity in vitro. and 10 g/L of trisodium citrate pentahydrate, adjusted to pH 7 with KOH. 9. Growth medium, e.g. commercial rich medium (2xYT or LB), or minimal medium. For 25 mL of minimal MD medium, mix 2.5 mL 10X PC buffer, 0.125 mL of 2.2 mg/mL ferric ammonium citrate CAF, 0.075 mL of 1M MgSO 4 and 1.25 mL of 40 mg/mL potassium aspartate (adjusted to pH 7 with KOH), 2% (w/v) carbon source (e.g. glucose or malate), and 0.25 mL of 5 mg/mL L-tryptophan for B. subtilis Trp auxotrophs. 10. B. subtilis strain of interest (our laboratory strain is the W168 strain SSB1002 [4]).

Materials

RNA decay experiments

Isolation of RNA from small culture volumes

All materials and solutions must be RNase free and gloves should be worn during the whole procedure. 16. 10 M LiCl in sterile, deionized water. 17. Acid-washed glass beads (150-220 µm diameter).

18. RNA extraction solution (for RNAsnap TM ): 95 % (v/v) Formamide, 18 mM EDTA, 0.025% (w/v) SDS, 1% (v/v) 2-mercaptoethanol. Prepare right before use (150 μL per sample).

Northern Blotting

All tubes and solutions must be RNase-free.

General

1. Water bath and thermostated dry temperature block.

2. Power supply for gel electrophoresis or electrotransfer.

3. Commercial high range or low range RNA markers for agarose and polyacrylamide gels, respectively.

4. Hybridization oven with a rotating rack for cylindrical glass hybridization bottles. 11. 1X TBE running buffer: Prepare 1L solution by dilution of 100 ml of 10X TBE stock with 900 ml of RNase-free water.

Polyacrylamide gels (5%)

1. Gel electrophoresis system (OmniBLOT, Cleaver scientific, or equivalent) with 20x20 cm glass plates, 1 mm spacers and 24-well gel comb for polyacrylamide gel electrophoresis.

2. Trans-Blot cell (BioRad), or equivalent, for polyacrylamide gel transfer.

3. 5% acrylamide solution: For 1 L, mix 420 g of urea with 100 mL of 10X TBE buffer and 125 mL of 40% acrylamide:bis-acrylamide solution (19:1). Complete to 1 L with sterile, deionized water. Put the solution on a magnetic stirrer for at least 1 hour, until the urea is completely dissolved. Filter the solution with a 0.2 µm pore-size filter. The solution can be stored at 4°C for several months in a dark bottle protected from light.

4. Silanization solution to pre-treat front gel plate (Sigma).

5. 10% (w/v) ammonium persulfate (APS) in water. Make 1 ml aliquots and store at -20 °C. Store at 4°C in 100 mL aliquots.

Radiolabeling of oligonucleotide probe

1. 10 mM Oligodeoxyribonucleotide complementary to mRNA (25 to 30-mer) in deionized water.

2. 10U/µL T4 Polynucleotide kinase (PNK) with supplied 10X PNK reaction buffer.

3. 3000 Ci/mmol γ 32 P-ATP.

G50 Microspin column (GE Healthcare).

5. Screens and protective clothing to work with 32 P.

Methods

RNA decay experiments

Rifampicin is a broad-spectrum antibiotic used principally to treat tuberculosis. It inhibits initiation of transcription by binding the -subunit of DNA dependent RNA polymerase (RNAP), allowing us to measure the rate of decay of RNA as a function of time, without interference from new transcription. If the stability of an mRNA is unknown, we typically isolate total RNA at several short and long time points (e.g. 2, 4, 6, 10, 20, and 30 min) after the addition of rifampicin to growing cultures in Erlenmeyer flasks. This can be adjusted to more appropriate time points once an initial decay pattern has been revealed. For practical reasons (i.e. it takes several seconds for rifampicin to mix evenly into the culture), our zero time point 'T0' actually corresponds to a sample taken 1 minute before the addition of rifampicin, with an assumption that relative amount of the mRNA of interest to all others will not significantly evolve in steady state growth during that minute (i.e. that T-1 = T0). The RNAs are probed by Northern blot (see below) for the specific mRNA of interest. The halflife (T½) of specific mRNAs is calculated by plotting the log of the percent remaining mRNA against time, with the initial amount of RNA 'T0' set at 100%. The slope of a linear regression of the data points on this graph is then used to calculate the half-life using the following formula: T½ = log2/-slope An example is shown in Figure 1. [Figure 1 near here] Note that for some mRNAs, the decay curve will be biphasic rather than linear, usually signifying two conformations/populations of the RNA in the cell. Thus, a judgement needs to be made for which data points it is more pertinent to measure the half-life. For stable mRNAs, measurement of half-life is not useful, as it usually surpasses the doubling time of the strain. However, a rifampicin kinetics experiment can be useful to follow the evolution of precursor species or decay products.

1. Prepare an appropriate number of 1.5 ml microtubes (1 for each time point) containing 100 µl of 10 mM sodium azide and freeze at -20°C.

2. Inoculate 5 ml of the specific medium of interest (e.g. 2xYT, LB or MD) with the B. subtilis strain of interest. Grow overnight at 37°C under shaking (200 rpm).

3. Use the overnight preculture to inoculate 15 mL of same medium culture (which is enough to measure the optical density at 600 nm [OD 600 ] regularly and to harvest for each data point) in 150 mL Erlenmeyer flasks at an OD 600 of 0.05. Incubate at 37°C with shaking at 200 rpm and regularly check OD 600 (see Note 1).

4.

As the OD 600 approaches the required value (e.g. mid-log ≈ 0.6 for B. subtilis), put the Microtubes containing frozen sodium azide on dry ice except for the 'T0' tube, which is put directly on ice. At T-1, pipette a 1-1.4 mL aliquot of culture on top of frozen sodium azide. Return flask immediately to orbital shaker at 37°C. Invert tube/vortex the sample tube until sodium azide has thawed, killing bacterial metabolism and reducing the temperature in one step (see Note 2).

5. 1 minute later, add rifampicin to a final concentration of 150 µg/ml (see Note 3) and immediately return flask to shaker. 6. Finish treating the 'T0' sample by centrifuging 1 minute at 16,000 g at 4°C.

Remove supernatant and put the pellet immediately in dry ice (see Note 4).

8. Repeat steps 6 and 7 for each time point after addition of rifampicin (T1, T2, T3, etc.) transferring the next azide-containing Microtube in the series from dry ice to ice just before harvesting (see Note 2).

Isolation of RNA from small culture volumes.

The following two protocols have been optimized to extract RNA from small culture volumes (1 -4 mL). These procedures are the same whether used on bacterial pellets harvested during a rifampicin kinetics experiment or from untreated cultures, e.g to compare RNA profiles in bacterial strains harvested at the same OD or between wt and mutant strains. Phenol extraction of RNAs from cells is relatively slow (4 h), but the samples are pure and can be used for both Northern blots (below) and enzymatic reactions, such as primer extension analysis. The RNAsnap TM procedure [5] is quick (30 minutes) but the presence of 95% formamide in the final sample means that they can only be used for Northern blotting. Note that the presence of formamide in the sample also affects the migration of RNA samples in the 200-300 nt range on 5% polyacrylamide gels, altering migration position and significantly reducing band sharpness.

1. For extraction of RNA, harvest up to 4 x 1 mL bacteria cells in 1.5 mL microtubes, depending on anticipated abundance of the mRNA in question. Centrifuge 2 min at 16,000 g at 4°C.

2.

Resuspend each 1 mL pellet in 100 μL ice cold TE buffer (see Note 5). If several pellets have been harvested, pool samples and re-centrifuge for 2 min at 16,000 g at 4°C.

Remove the supernatant and keep pellets in dry ice or store at -20°C until RNA extraction. The Northern blot technique allows the detection of a specific RNA species among total RNA run on gels and is used both to quantify the amounts of the RNA of interest in a given (environmental, mutant) condition, and to determine the number and sizes of the different RNA species transcribed from a particular locus. This method therefore has major advantages over other techniques for quantifying RNA, such as qRT-PCR, in particular because the experimenter can immediately assess the quality of the RNA preparation and tell whether it is degraded or not. Depending on the anticipated size of the RNA being studied, total RNA is separated on agarose gels (typically 1% agarose for the detection of RNAs >300 nucleotides

Proceed to step 4 for extraction by the RNAsnap

[nts]) or on polyacrylamide gels (typically 5% acrylamide for RNA molecules <300 nts).

RNAs are transferred to nylon membranes by capillarity or electrophoretically, respectively.

We describe here the two types of Northern blot (agarose and polyacrylamide) to detect RNAs with radioactive probes, which are typically an order of magnitude more sensitive than fluorescently labelled probes, for example.

Gel electrophoresis and membrane transfer

1. For detection of RNAs > 300 nts, proceed to step 2 for agarose gels. For longer RNAs, proceed to step 12 for polyacrylamide gels. 21. On the negative grid of the transfer cassette, add a sponge soaked in 0.5X TBE buffer. 2. For data points that are close together, it is advisable to find a lab partner to run the centrifuge, remove supernatants and freeze samples, while the other adds rifampicin, samples the cultures and mixes with frozen azide. 8. Work under a chemical fume hood. 9. Be careful not to dry the pellet too much because it will be difficult to resuspend. 10. After transfer, RNAs can be visualized with a portable UV light at 254 nm. Mark the ribosomal RNAs and the bands of the RNA marker with a pencil, pressing strongly. This will score the membrane and hold the radioactivity during the hybridization, allowing the visualization of the marker without its labelling.

11. Silanization of the plate should be done under a fume hood (only 1 plate should be treated!) 12. Use a plastic tool to avoid damaging the glass plates. 20 13. Dry the Whatman 3MM paper with paper towel to remove liquid excess and allow the gel to stick to the Whatman 3MM paper.

14. If the RNA of interest is expressed at a very low level, the radiolabeled oligonucleotide can be replaced by an α-32P -UTP labelled riboprobe complementary to the RNA. This will often improve the signal, but note that it is difficult to remove if the membrane is to be reprobed for another RNA. We usually make riboprobes between 200 and 500 nucleotides in length, using a PCR template with a T7 promoter integrated into the lower oligo (see chapter Y). The pre-hybridization and hybridization temperatures should be increase to 68°C if a riboprobe is used instead of radiolabeled oligonucleotide.

15. All radioactive materials should be handled by properly trained scientists, following safety rules and local regulations.

5 .

 5 UV lamp (254 nm) for cross-linking. The UVP HL-2000 Hybrilinker (UVP) has both oven and cross-linker functions. 6. Benchtop microcentrifuge. 7. Geiger counter. 8. Pencil. 9. PhosphorImager (Typhoon, GE Healthcare) with dedicated 32 P phosphorimaging screens and image analysis software. 10. 10X Tris-Borate-EDTA (TBE) buffer. Buy RNase-free stock from reliable commercial source.

  6. N,N,N',N'-Tetramethylethylenediamine (TEMED).

  7. 0.5X TBE buffer, for electrophoretic transfer buffer. Prepare 4 L solution by dilution of 200 ml of 10x TBE buffer in 3.8 L of RNase-free water. Store at 4°C. 8. Roti-Hybri-Quick (Roth) for acrylamide Northern blots. Denature 20 mL of the 20 mg/mL stock solution herring or salmon sperm DNA and add to 1L Roti-Hybri-Quick.

  TM procedure, or to step 12 for extraction by phenol. A maximum of 12 samples can be extracted at a time with the Genie Cell Disruptor 4. Fill empty 1.5 mL microtubes to 100 μL mark with glass beads (one tube per sample).

2 .

 2 Prepare a 1% agarose gel by weighing 1 g of agarose and transfering to an Erlenmeyer flask. Add 100 mL of 1X TBE. Bring to the boil in a microwave. Remove from microwave, mix by swirling, bring back to boil 2-3 times until all agarose is solubilized. Cool solution until temperature is between 40° and 50°C. After cooling, add 6 µL of 1% ethidium bromide solution and pour the gel in a dedicated mold. Add the gel comb and allow gel to set for 1 hour. 3. Mix 5 µg of total RNA with an equal volume of gel loading buffer. The final volume should not exceed 20 µL. 16. Rinse the gel wells with a Pasteur pipette filled with 1X TBE to remove urea and polyacrylamide fragments. 17. Mix 5 µg of total RNA with an equal volume of gel loading buffer. The final volume should not exceed 20 µL.

18 .

 18 Denature RNAs by heating samples for 5 min at 90°C. Rinse the gel wells rapidly again with a Pasteur pipette and then load RNA samples in the wells. 19. Run the gel at room temperature at 400V. Stop the migration when the bromophenol blue dye reaches about 1 cm from the bottom of the gel. 20. Separate the gel plates with the help of a spatula or a similar tool (see Note 12).

22 .

 22 Soak a piece a 3MM Whatman paper equal to the size of the gel (20 cm x 20 cm) in 0.5X TBE and place it on the sponge. 23. Soak a second 3MM Whatman paper equal to the size of the gel (20 cm x 20 cm) in 0.5X TBE and apply to the gel still attached to the glass plate. Optimize contact with the gel and remove excess liquid by blotting with a paper towel (see Note 13). 24. Peel the paper with the gel stuck to it from the glass plate and place it (gel facing up) on the first piece of Whatman 3MM paper. 25. Soak the Hybond N+ membrane equal to the size of the gel in 0.5X TBE and place it on the gel. Use a pipette or glass rod to roll out air bubbles. 26. Add two more sheets of 3MM Whatman paper pre-soaked in 0.5X TBE on the membrane, rolling out air bubbles. 27. Add a second sponge soaked in 0.5X TBE and close the transfer cassette. 28. Place the cassette in the BioRad Trans-Blot in the correct orientation (membrane towards positive pole) and fill the tank with 0.5X TBE precooled at 4°C. 29. Transfer for 2 hours at 250 mA and an additional 2 hours at 350 mA (see Note 10).

3 .

 3 Rifampicin is light sensitive and stored resuspended in DMSO or DMF in foil covered tubes; wear gloves during manipulation.4. For minimal medium (e.g. MD or M9), it is important to wash the pellet by resuspending in 1 mL of TE/NaCl buffer to remove excess phosphate and to centrifuge a second time before freezing the pellet on dry ice. 5. When working with RNAs be careful to avoid any contamination with RNases. Wear gloves at all times, and prior to your experiments clean your bench and your pipettes with water then ethanol or RNase away solution. We keep a separate set of micropipettes reserved for RNA work.6. OD 260 /OD 280 ratios are generally poor for RNAsnap TM preps 7. Samples should be equilibrated at room temperature before measurement of RNA concentration to avoid interference from the SDS present in the solution that precipitates in the cold.

Figure 1 :

 1 Figure 1: Example of half-life measurement.

Figure 2 :

 2 Figure 2: Schematic of capillary transfer set up for agarose gels

Acknowledgments

This work was supported by funds from the CNRS (UMR8261), Université de Paris, the Agence Nationale de la Recherche (ARNr-QC; BaRR) and the Labex (Dynamo) program.