植物核糖体的足迹分析实验

Catharina  Merchante; Qiwen  Hu; Steffen  Heber; Jose Alonso; Anna  N. Stepanova

doi:10.21769/BioProtoc.1985

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Peer-reviewed

A Ribosome Footprinting Protocol for Plants

植物核糖体的足迹分析实验

Anna N. Stepanova email

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1985 浏览次数: 14288

评审: Samik BhattacharyaRenate WeizbauerBaohua Li

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Abstract

Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid culture [unpublished results]).

Background

The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. The ribosome footprinting technology (often known as the Ribo-seq), developed originally for yeast and mammalian cells in culture, has revolutionized the studies of translation regulation and gene expression, as it allows to determine the exact positions of the ribosomes at a genome-wide scale and at a single-codon resolution (Ingolia et al., 2009).

Prior to the development of Ribo-seq, the most common methods employed to study translation regulation in plants were the isolation of polysomal RNA via sucrose gradient centrifugation or translating ribosome affinity purification (TRAP) followed by Northern blotting, qRT-PCR, microarrays, or RNA-seq. The first method, known as polysome profiling, relies on resolving distinct polysomal fractions on a sucrose gradient via ultracentrifugation (Branco-Price et al., 2008; Missra and von Arnim, 2014; Li et al., 2015). By comparing different plant growth conditions or mutants, one could infer the changes in the rates of translation from observing a shift in the distribution of mRNAs between polysomal fractions. For example, if a transcript becomes more abundant in the monosomal fraction with the concomitant decrease in the higher order polysomes, the translation of this mRNA is considered as down-regulated. The key limitation of this technique, however, is its low resolution of higher-order polysomes (and thus mild quantitative changes in translation are often missed) and the inability to differentiate between polysomal RNAs that undergo active translation versus are loaded with arrested ribosomes (for example, those ‘stuck’ in the upstream open reading frames of a transcript). The second polysomal RNA isolation technique, TRAP, is based on the stable expression of an epitope-tagged ribosomal protein followed by the immunoprecipitation of entire ribosomes along with their associated mRNAs (Zanneti et al., 2005; Reynoso et al., 2015). While this latter method accommodates both global and tissue-specific studies of translation (achieved by driving the expression of a tagged ribosomal protein in a ubiquitous versus tissue-specific manner), its use is limited to transformable species where transgenic lines can be generated. Furthermore, transcriptomic analysis of TRAP samples per se does not provide a quantitative measure of translation (unless coupled with Ribo-seq [Juntawong et al., 2014]), as any mRNA with one or more ribosomes bound to it will be purified by TRAP. Also, since TRAP relies on epitope-tagging and the tag may interfere with the function of the ribosome, the regulation of translation of some mRNAs may be disrupted in the TRAP transgenic lines, e.g., due to a reduced ability of the tagged ribosome to associate with specific proteins at certain stages of translation. Another limitation of TRAP is that it typically uses a specific redundant isoform of a ribosomal protein for tagging, such as RPL18, and thus likely purifies only a subset of ribosomes that carry just that RPL18 variant. Given that there are multiple RPL18-like proteins in plant genomes, using one specific ribosomal protein isoform for tagging misses the ribosomes that utilize an alternative RPL18 isoform.

The method of choice for our studies, the Ribo-seq, does not involve transgenic line generation nor affinity purification, thus avoiding many of the limitations of the aforementioned earlier techniques. Most importantly, the single-codon resolution of the ribosome footprinting technology allows researchers to map the ribosomes on the mRNAs and thus clearly distinguish between the transcripts harboring productive ribosomes translating the main genic open reading frames versus transcripts associated with non-productive ribosomes arrested in the 5’UTRs. Not only does this method offer a snapshot of a whole-genome view of ribosomal distribution at an unprecedented resolution, it also enables the true quantitative measure of translational efficiency of every expressed gene in the genome by correlating the Ribo-seq data with the transcriptional information obtained via RNA-seq. Nonetheless, even the Ribo-Seq has its own drawbacks, as it cannot discriminate between mRNA subpopulations with different translation efficiencies, giving an average translation efficiency readout for each expressed gene.

Herein, we provide a plant-optimized Ribo-seq protocol that enables the study of translation regulation through the isolation of high-quality ribosomal footprints from different developmental stages of in vitro grown Arabidopsis thaliana seedlings. It describes step by step how to pellet and digest polysomes, isolate monosomes, extract the mRNA footprints, and generate sequencing libraries for the Illumina platform. The protocol also describes the preparation of parallel RNA-seq libraries to account for transcriptional regulation. We conclude the description of our method with a brief summary of how to analyze the sequencing results.

Materials and Reagents

Materials
1. Miracloth
2. 30 ml Nalgene^TM High-Speed polycarbonate centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3118-0030 ) (or equivalent) to centrifuge the plant extracts
3. 5 ml polypropylene thin-wall ultracentrifuge tubes (Beckman Coulter, catalog number: 326819 )
4. 10 ml ultracentrifuge 14 x 89 mm tubes with isopycnic caps (BioComp Instruments, catalog number: 105-914A )
5. Fine scale
6. 10 ml syringe
7. Cannula to be attached to the syringe (Thomas Scientific, catalog number: 1193G13 )
8. Pre-sterilized, RNase-free 5 ml, 1 ml, 200 µl and 10 µl micropipette tips
9. Pre-sterilized, RNase-free 2 ml and 1.5 ml microcentrifuge tubes
10. 15 and 50 ml Falcon tubes
11. Stoppers to secure the lids of 1.5 ml tubes
12. Razor blades
13. Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Ambion^TM, catalog number: 610-06 )
14. 2 ml microcentrifuge tubes
15. 1.5 ml non-stick, RNase-free microtubes (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: AM12450 )
16. MyOne^TM Streptavidin C1 DynaBeads^® (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 65601 )
17. 200 μl PCR strip tubes
Reagents
1. Liquid nitrogen
2. RNase-free, sterile, MilliQ water
3. Tris base
4. Sucrose
5. Potassium chloride (KCl)
6. Sodium chloride (NaCl)
7. Magnesium chloride (MgCl₂)
8. Ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N-tetraacetic acid (EGTA)
9. Ethylene diamintetracetic acid (EDTA)
10. Dithiothreitol (DTT)
11. Brij-35 [Polyoxyethylene(23)lauryl ether]
12. Triton X-100
13. Igepal CA 630 (Octylphenyl-polyethylene glycol)
14. Tween 20 (Polyoxyethylene sorbitan monolaurate 20)
15. Cycloheximide (see Note 1)
16. Chloramphenicol (see Note 1)
17. Lithium chloride (LiCl)
18. Sodium acetate (NaOAc)
19. Sodium bicarbonate (NaHCO₃)
20. Sodium carbonate (Na₂CO₃)
21. Sodium dodecyl sulfate (SDS)
22. Sodium hydroxide (NaOH)
23. Trisodium citrate (Na₃C₆H₅O₇)
24. Dimethylsulfoxide (DMSO), PCR grade
25. Ethanol, molecular biology grade
26. Water-saturated, acid phenol, molecular biology grade (see Note 2)
27. Chloroform, molecular biology grade (see Note 2)
28. Isoamyalcohol
29. Isopropanol, molecular biology grade
30. PEG8000
31. 15 mg/ml GlycoBlue^TM (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9515 )
32. 10 bp DNA ladder (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10821015 )
33. 2x denaturing sample buffer with dye (Thermo Fisher Scientific, Invitrogen^TM, catalog number: LC6876 ) (see Note 3)
34. SYBR^® Gold, 10,000x in DMSO (Thermo Fisher Scientific, Invitrogen^TM, catalog number: S11494 ) (see Note 4)
35. dNTP mix, 10 mM (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18427-013 )
36. OmniPur^® polyethylene glycol 8000 (EMD Millipore, Calbiochem^®, catalog number: 6510 )
37. Universal miRNA cloning linker (New England Biolabs, catalog number: S1315S )
38. Boric acid
39. Acetic acid
40. 12-well 15% polyacrylamide TBE-Urea gels (Bio-Rad Laboratories, catalog number: 4566055 ) (see Note 5)
41. 30% acrylamide/bisacrylamide (29:1) (Bio-Rad Laboratories, catalog number: 161-0156 ) (see Note 5)
42. Ammonium persulfate (APS)
43. TEMED (N,N,N’,N’-Tetramethylethane-1,2-diamine) (Sigma-Aldrich, catalog number: T7024 )
44. Glycerol
45. Bromophenol blue
Enzymes
1. SUPERase-In^TM RNase inhibitor (Thermo Fisher Scientific, Ambion^TM, catalog number: AM2694 )
2. TURBO^TM DNase (2 U/μl) (Thermo Fisher Scientific, Ambion^TM, catalog number: AM2238 )
3. RNase I (100 U/μl) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM2294 )
4. SuperScript^® III reverse transcriptase (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18080-093 )
5. T4 polynucleotide kinase, T4 PNK4 (New England Biolabs, catalog number: M0201 ), supplied with 10x T4 PNK buffer (New England Biolabs, catalog number: B0201 ) (see Note 6)
6. T4 RNA ligase 2, truncated (New England Biolabs, catalog number: M0242 ), supplied with PEG 8000 50% and 10x T4 Rnl2 buffer
7. Phusion high-fidelity DNA polymerase (2 U/μl) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: F-530S )
8. CircLigase^TM ssDNA ligase (100 U/μl) (Epicentre, catalog number: CL4115K )
Oligonucleotides
1. Reverse transcription primer for split-adapter circularization (see Note 7)
  NI-NI-9: [P]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC[SC18]CACTCA[SC18]TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG (Ingolia et al., 2009)
2. Size marker oligos (Ingolia et al., 2009) (see Note 8)
  oNTI265: rArUrGrUrArCrArCrGrGrArGrUrCrGrArGrCrUrCrArArCrCrCrGrCrArArCrGrCrGrA
  oNTI268: rArUrGrUrArCrArCrGrGrArGrArCrCrCrGrCrArArCrGrCrGrA
3. rRNA subtraction oligos (all the oligos here have a 5’ TEG-linked biotin and were obtained from IDT),These oligos are based on the most abundant rRNA sequences and transposons that were found using 3-day-old Arabidopsis seedlings and the ribosome footprinting protocol described herein (see Note 9)
  rRNABio1: 5’-gataaccgtagtaattctagag-3’
  rRNABio2&4: 5’-TGATTCATGATAACTCGACGGACGACGCGGATTACGGTGGCGGC-3’
  rRNABio5&3: 5’-GTCGCTGCCGTGATCGTGGTCTCCATCGAGTCTTTGAACGCAAG-3’
  Bio-Tranpos: 5’-GAGGGATGCAACACGAGGAGTTCCCGGGAGGTCA-3’
  Bio-5S: 5’-AAGCCTTCTGGCCGAGGGCACGTCTGCCTGGGTGTCACAA-3’
  Bio-18S: 5’-AAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTG-3’
4. Amplification primers
  NI-NI-2: 5’-AATGATACGGCGACCACCGAGATCTACAC-3’ (Ingolia et al., 2009)
  NI-NI-3: 5’-CAAGCAGAAGACGGCATACGAGATAGTCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’ (Ingolia et al., 2009)
  Alternative indexed primers
  Index 1:
  5’-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 2:
  5’-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 3:
  5’-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 4:
  5’-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 5:
  5’-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 6:
  5’-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
  Index 7:
  5’-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCG-3’
Solutions
All solutions should be prepared with RNase-free, MilliQ water. Unless otherwise stated, all of them must be sterilized by autoclaving for at least 20 min and stored at room temperature.
1. 1 M Tris-HCl, pH 7.0 (see Note 10)
2. 1 M Tris-HCl, pH 8.0 (see Note 10)
3. 1 M Tris-HCl, pH 9.0 (see Note 10)
4. 1 M sucrose, prepared from Molecular Biology grade sucrose (autoclave for 10 min)
5. 1 M KCl
6. 5 M NaCl
7. 1 M MgCl₂
8. 0.5 M EGTA, pH 8.0 (see Note 11)
9. 0.5 M EDTA, pH 8.0 (see Note 11)
10. 2 mM EDTA, 100 mM Na₂CO₃
11. 2 mM EDTA, 100 mM NaHCO₃
12. 3 M NaOAc, pH 5.5
13. 10% SDS (w/v)
14. 1 M NaOH (no sterilization required)
15. 4 M LiCl (filter-sterilize, do not autoclave)
16. 1 M DTT (do not autoclave; aliquot and store at -20 °C)
17. 100 mg/ml cycloheximide, prepared in DMSO (filter-sterilize; aliquot and store at -20 °C)
18. 50 mg/ml chloramphenicol, prepared in ethanol (filter-sterilize; aliquot and store at -20 °C)
19. Detergent mix 20% (w/v or v/v) of each of four detergents in water (Brij-35, Triton X-100, Igepal CA 630 and Tween 20)
20. 50% PEG8000 (w/v), prepared by combining dry PEG with water in a 50 ml conical and mixing with gentle shaking overnight. No sterilization is required. PEG is very hygroscopic; so start adding PEG to less than half of the final volume of MilliQ water. Then, after it is dissolved, add more water if needed. PEG used for ligation (step C3) should not be more than 1 month old.
21. 10% APS (w/v), Filter-sterilize and aliquot in small volumes (e.g., 500 µl). Store at -20 °C. Aliquots can be thawed and re-frozen several times.
Buffers (see Recipes)
1. Polysome extraction buffer (PEB)
2. Sucrose cushion A (SCA)
3. Sucrose gradients solutions
4. Polysome digestion buffer (PDB)
5. Sucrose cushion B (SCB)
6. Polysome resuspension buffer (PRB)
7. Total RNA extraction buffer (TREB)
8. Alkaline fragmentation buffer (2x)
9. Alkaline fragmentation stop/precipitation solution
10. TAE (Tris/acetate/EDTA buffer) (50x)
11. TBE (Tris/borate/EDTA buffer) buffer (5x)
12. RNA gel extraction buffer (GEB)
13. DNA gel extraction buffer (STE)
14. SSC (20x)
15. Subtraction bind/wash buffer (2x)
16. 8% non-denaturing polyacrylamide gel (12 ml)
17. Non-denaturing loading dye (6x)

Equipment

7-9 cm diameter porcelain mortar and pestle
Small (30-50 ml) glass beakers
Refrigerated Beckman Avanti J-25 centrifuge and Beckman JA17 rotor (or equivalent)
Ultracentrifuge Beckman L8-70M (or equivalent) with swinging bucket rotors for polysome pelleting (Beckman Coulter, model: SW55Ti ) and sucrose gradient centrifugation (Beckman Coulter, model: SW41Ti or Thermo Fisher Scientific, Thermo Scientific^TM, model: TH-641 ).
Automatic P1000, P200 and P10 micropipettes
Refrigerated tabletop microcentrifuge
Orbital shaker
Gradient Master Station, with both the gradient maker and the fractionation station (BioComp Instruments)
Continuous UV light monitor (Bio-Rad Laboratories, model: EM-1 Econo UV Monitor )
Fractionator (Gilson, model: FC203B )
Nanodrop (or another spectrophotometer to quantify nucleic acid concentrations in small volumes of RNA)
Nutator shaker
Thermoblock
Fume hood
Mini-PROTEAN tetra cell polyacrylamide gel box (Bio-Rad Laboratories, catalog number: 165-8004 ) or equivalent and electrophoresis power supply
UV transilluminator
Vortex
DynaMag^TM-2 magnetic separation rack (Thermo Fisher Scientific, catalog number: 12321D )
Thermocycler
2100 BioAnalyzer (Agilent Technologies, catalog number: G2940CA )
HiSeq2000 (Illumina)

Procedure

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Merchante, C., Hu, Q., Heber, S., Alonso, J. and Stepanova, A. N. (2016). A Ribosome Footprinting Protocol for Plants. Bio-protocol 6(21): e1985. DOI: 10.21769/BioProtoc.1985.