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. Author manuscript; available in PMC: 2021 Jun 30.
Published in final edited form as: Methods Mol Biol. 2021;2298:105–122. doi: 10.1007/978-1-0716-1374-0_7

miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling

Vanja Stojković 1, David E Weinberg 1, Danica Galonić Fujimori 2,3
PMCID: PMC8245296  NIHMSID: NIHMS1714016  PMID: 34085241

Abstract

The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.

Keywords: RNA methylation, Radical SAM, Substrate identification, Methyl adenosine, RlmN, Cfr, TGIRT

1. Introduction

There are more than 100 chemically distinct RNA modifications, out of which methylation is the most common. RNA methylation is ubiquitous across all domains of life; however, the exact location, biological function, and corresponding RNA-methylating enzymes are poorly understood. Recent strategies that combine immunoprecipitation of modified RNA or chemical treatment of RNA with next-generation sequencing have allowed mapping of the location and abundance of a subset of RNA modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ) [1–8]. These approaches take advantage of either the unique chemical reactivity of the methyl group (e.g., detection of m5C via RNA bisulfite sequencing) [3] or the availability of modification-specific antibodies (e.g., transcriptome-wide identification of m6A and Ψ) [1, 4, 9]. Additionally, strategies based on UV cross-linking and immunoprecipitation, known as CLIP-based methods, have been used to identify enzyme-substrate pairs for RNA-modifying enzymes [10–14]. These methods employ UV irradiation to generate covalent protein-RNA adducts that can be subsequently isolated and enriched to identify RNA-interacting partners. Despite many advances that resulted from CLIP-based methods, the main disadvantage of UV cross-linking is its low cross-linking efficiency. An alternative approach developed for substrate identification for some RNA methyltransferases exploits the formation of a covalent catalytic intermediate between the enzyme and its RNA substrate [3, 15]. While inherently limited to enzyme families that form a covalent intermediate in their catalytic mechanisms, this approach allows for highly efficient cross-linking of enzyme-substrate pairs. This strategy has earlier been applied to NSun RNA methyltransferase family members, which methylate cytosines in RNA to yield m5C. The covalent enzyme-substrate intermediate is trapped either by using a 5-azacitidine (Aza) analog, as in Aza-IP [3], or by mutation of the key cysteine residue in these enzymes that is necessary for the resolution of the covalent intermediate, as in methylation-iCLIP (miCLIP) approach [15].

Radical SAM RNA-methylating enzymes employ a radical-based mechanism to generate 2-methyladenosine (m2A, RlmN enzymes) and 8-methyladenosine (m8A, Cfr enzymes). Mechanistic studies by our group [16] and others [17–20] have revealed that substrate methylation by radical SAM RNA-methylating enzymes proceeds through an enzyme-substrate covalent intermediate distinct from those formed by RNA m5C methyltransferases [21]. The hallmark of the methylation is formation of a methylene-bridged covalent intermediate between a Cys residue in the enzyme (C355 in E. coli RlmN) and amidine carbon of the adenosine substrate (Fig. 1) [16–20, 22, 23]. Subsequently, the enzyme-RNAcovalent adduct is resolved by a second conserved cysteine (C118 in E. coli RlmN) [16]. Mutation of C118 (C118A) stabilizes the protein-RNA intermediate, enabling isolation of the enzyme-RNA covalent pairs by immunoprecipitation (Fig. 1) [16]. By combining this key mechanistic feature with next-generation sequencing, we have developed a novel strategy where individual-nucleotide-resolution cross-linking and immunoprecipitation are combined with mutational profiling with sequencing (miCLIP-MaPseq) [24]. This method can allow for the identification of substrates and modification sites for any member of the radical SAM RNA-methylating enzyme family. The method was developed and validated using the most well-characterized member of the family, RlmN from E. coli that is known to modify 23S rRNA, as well as a subset of tRNA substrates [25, 26].

Fig. 1.

Fig. 1

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Mechanistic scheme for RlmN-mediated methylation of RNA showing key steps. The stable covalent intermediate trapped by C118A mutation is shown

miCLIP-MaPseq relies on immunoprecipitation of a stable covalent complex between the mutant enzyme and RNA, followed by high-throughput RNA sequencing (Fig. 2). Following isolation, the protein-RNA species are digested using Proteinase K, which leaves a peptide scar on the RNA at the site of the protein-RNA cross-link formation. RNA is then size-selected on a denaturing TBE-urea gel (Fig. 3). RNA species larger than 300 nucleotides are fragmented prior to dephosphorylation [27], while smaller RNA fragments are dephosphorylated without prior fragmentation. After size selection, RNAs are converted to cDNA using the TGIRT reverse transcriptase, and the resulting library is subjected to PCR amplification and high-throughput sequencing. One main advantage of miCLIP-MaPseq is that it uses TGIRT to generate cDNA. This reverse transcriptase is highly processive and introduces a mismatch when it encounters the protein scar on RNA and thus allows identification of methylation sites using mutational profiling. Here, we provide a detailed miCLIP-MaPseq protocol that can be easily modified and implemented to identify substrates of any member of the radical SAM RNA-methylating family.

Fig. 2.

Fig. 2

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Schematic representation of library preparation strategy for identification of substrates and methylation sites of RlmN. Red bars represent the fraction of mismatches at a specific nucleotide on substrate RNA

Fig. 3.

Fig. 3

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Gel analysis of isolated RNA after immunoprecipitation and Proteinase K treatment of FLAG-tagged C118A RlmN. RNA was size selected into four regions (A-D) as indicated on the gel, and each region was individually sequenced. Lanes 1–3: Isolated RNA after immunoprecipitation and Proteinase K treatment of FLAG-tagged C118A RlmN, where the amount of sample loaded in lane 1 is half of the amount loaded in each of the lanes 2 and 3; lane 4: low-range single-stranded RNA markers

2. Materials

2.1. Cell Lysis and Target Protein Immunoprecipitation

  1. Lysis buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100.

  2. 100 mM PMSF.

  3. TBS buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl.

  4. Glycine buffer: 100 mM glycine–HCl pH 3.5.

  5. Stringent-TBS wash: 50 mM Tris–HCl pH 7.5, 500 mM NaCl.

  6. Resin recycle solution: 50% Glycerol, 50% TBS, 0.02% sodium azide.

  7. 10 mM Tris pH 7.5.

  8. RQ1 RNase-free DNase.

  9. Anti-FLAG M2 resin.

  10. IP dilution solution: 12 μL of 5 mg/mL 1× FLAG peptide and 388 μL of 10 mM Tris pH 7.5.

  11. LB medium.

  12. 1.5 mL Eppendorf tubes.

  13. E. coli strain encoding a FLAG-tagged wild-type RNA-modifying enzyme of interest and the corresponding FLAG-tagged mutant RNA-modifying enzyme. For the example described herein, use E. coli BW25113 strain encoding FLAG-tagged wild-type RlmN and E. coli BW25113 FLAG-tagged C118A RlmN.

2.2. RNA Isolation

  1. Proteinase K.

  2. GlycoBlue.

  3. 3 M Sodium acetate pH 5.5.

  4. Isopropanol.

  5. 80% Ethanol.

  6. Novex 10% TBE-urea precast gel.

  7. 1× TBE running buffer: Dilute 10× TBE running buffer to 1× using DEPC-treated water.

  8. 2× RNA loading dye.

  9. Low-range ssRNA ladder.

  10. SYBR Gold Nucleic Acid Gel Stain.

  11. 18G × 1 ½ syringe needle.

  12. Costar SpinX column.

  13. Nuclease-free water.

  14. RNase-free non-stick 0.5 mL tubes and RNase-free 1.5 mL tubes.

  15. 100% Ethanol.

2.3. RNA Fragmentation

  1. 10× Fragmentation Reagent mix.

  2. Nuclease-free water.

  3. RNase-free PCR tubes.

2.4. Library Preparation

  1. T4 polynucleotide kinase.

  2. SUPERase-In.

  3. TGIRT-III template-switching kit.

  4. 10× PNK buffer: 70 mM Tris–HCl pH 7.5, 10 mM MgCl2, 5 mM DTT.

  5. 5× TGIRT reaction buffer: 100 mM Tris–HCl pH 7.5, 2.25 M NaCl, 25 mM MgCl2.

  6. Novex 8% TBE precast gel.

  7. 5× GelPilot DNA Loading Dye.

  8. 5′ DNA Adenylation Kit.

  9. Zymo Oligo Clean & Concentrator kit.

  10. Thermostable 5′ AppDNA/RNA Ligase.

  11. 10× NEBuffer 1.

  12. 50 mM MnCl2.

  13. MiniElute PCR Purification Kit.

  14. Phusion High-Fidelity DNA Polymerase.

  15. Deoxynucleotide (dNTP) Solution Mix.

  16. 10 mM Tris, pH 8.

  17. Oligos used for library preparation:
    • R2 RNA (provided in a kit by InGex; 3SpC3 is a C3 Spacer phosphoramidite): 5′-rArGrA rUrCrG rGrArA rGrArG rCrArC rArCrG rUrCrU rGrArArCrUrCrCrArG rUrCrA rC/3SpC3/-3′.
    • R2R DNA (provided in a kit by InGex): 5′-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TN (N = equimolar A, T, G, C).
    • R1R DNA (IDT; 3SpC3 is a C3 Spacer phosphoramidite): 5′-/5Phos/GAT CGT CGG ACT GTA GAA CTC TGA ACG TGT AG/3SpC3/-3′.
    • Illumina multiplex primer (IDT): 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C-3′.
    • Illumina barcode primer (IDT): 5′-CAA GCA GAA GAC GGC ATA CGA GAT [barcode] GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3′.

2.5. Library Quantification

  1. KAPA library quantification kit.

  2. 96-Well PCR plate.

  3. BioRad CFX Connect real-time PCR

  4. Agilent 2100 Bioanalyzer.

2.6. Sequencing

  1. Illumina HiSeq4000.

3. Methods

3.1. Expression of the FLAG-Tagged Enzyme

A C-terminal DYKDDDDK octapeptide (FLAG) sequence is fused to the genomic version of rlmN or rlmN containing C118A mutation as described previously [16].

  1. Inoculate 1 L of LB medium with 1:100 from an overnight culture of E. coli BW25113 encoding either the FLAG-tagged WT RlmN or the FLAG-tagged C118A RlmN.

  2. Grow the cells at 37 °C, 200 rpm, for 90 min for WT RlmN, or 150 min for C118A RlmN (see Note 1).

  3. Harvest the cells by centrifugation at 1610 × g (rotor F9S 4 × 1000y) for 15 min at 4 °C. Flash freeze the pellets in liquid nitrogen, and either store them at −80 °C or immediately proceed with the next step.

3.2. Lysis and DNase Treatment

  1. Thaw ~1.5 g of cells and resuspend them in 4 mL of cold lysis buffer. Add 40 μL of 100 mM PMSF.

  2. While keeping the cells on ice, sonicate the cells using the microtip on a power setting 3 and duty cycle 50%, for three 40-s pulses with 1-min breaks between pulses. The probe should be positioned approximately 0.5 cm from the bottom of the tube and should not be touching the tube sides in order to avoid foaming.

  3. Divide 4 mL sample among five 1.5 mL Eppendorf tubes. Add 18 μL RQ1 RNase-free DNase to each tube and incubate at 37 °C for 15 min. Spin down at 19,722 × g (Sorvall Legend Micro 21R) for 10 min at 4 °C. Without disturbing the pellet remove the supernatant to a new 1.5 mL tube. If not immediately proceeding with the next step, store the samples at −20 °C.

3.3. Immunoprecipitation

3.3.1. Resin Preparation

  1. Thoroughly suspend the anti-FLAG M2 affinity resin and immediately transfer 375 μL to a new Eppendorf tube. Centrifuge the resin at 8000 × g, for 1 min at 4 °C. Let the resin settle for 1 min. Remove the supernatant making sure not to transfer any resin.

  2. Wash the resin twice with 1 mL of TBS buffer. For each wash, add 1 mL of TBS buffer, resuspend the resin by gently pipetting, centrifuge the resin at 8000 × g for 1 min at 4 °C, and then let it settle for 1 min.

  3. Wash the resin once with 1 mL glycine buffer (see Note 2).

  4. Wash the resin three times with TBS buffer. For each wash, the process is the same as in Step 2.

3.3.2. Binding of FLAG-Tagged Protein

  1. Add approximately 75 μL of resuspended resin into each tube containing ~800 μL sample. Let the samples incubate with the resin on a rotator for at least 2 h at 4 °C.

3.3.3. Elution of FLAG-Tagged Protein

  1. Centrifuge the samples at 8000 × g, for 1 min at 4 °C. Let the resin settle for 1 min. Remove the supernatant.

  2. Wash the resin three times with 500 μL of stringent TBS wash buffer.

  3. Add 75 μL of IP dilution solution into each sample. Gently resuspend and rotate samples for at least 1 h at 4 °C (see Note 3).

  4. Centrifuge the samples at 8000 × g, for 1 min at 4 °C. Let the resin settle for 2 min.

  5. Carefully transfer the supernatant to a new Eppendorf tube. Put both resin and supernatant on ice. Store resin without glycerol at 4 °C. Store supernatant at −20 °C.

3.3.4. Recycling of the Resin

  1. Resuspend used resin in 500 μL of TBS buffer. Centrifuge the resin at 8000 × g, for 1 min at 4 °C. Remove the supernatant.

  2. Wash the resin three times with 1 mL of glycine buffer. Centrifuge the resin at 8000 × g, for 1 min at 4 °C. Let the resin settle for 1 min.

  3. Wash the resin five times with 1 mL of resin recycle solution. Centrifuge the resin at 8000 × g, for 1 min at 4 °C. Store the resin at −20 °C.

3.4. Proteinase K Treatment

  1. Thaw the sample obtained after immunoprecipitation on ice. Remove 10 μL for subsequent Western blot analysis. Add 9.6 U of Proteinase K into the rest of the sample and incubate the reaction for 2 h at 37 °C.

  2. Divide each sample into two Eppendorf tubes (~230–250 μL). Precipitate RNA by adding 1000 μL of isopropanol, 25 μL of sodium acetate pH 5.5, and 2 μL of GlycoBlue co-precipitant into each tube. Leave the tubes overnight at −20 °C.

  3. The next day precipitate RNA by centrifugation at 20,000 × g for 30–40 min at 4 °C.

  4. Carefully remove the supernatant and wash the pellet with 750 μL of 80% cold ethanol. Precipitate RNA by centrifugation at 20,000 × g for 40 min at 4 °C. Carefully remove the supernatant and air-dry the pellets (see Note 4).

  5. Resuspend pellets with 40 μL nuclease-free water (see Note 5).

3.5. Gel Purification and RNA Extraction

  1. Set up the gel apparatus and pre-run the 10% TBE-urea gel at 180 V for at least 20 min in 1× TBE running buffer.

  2. Add 25 μL of 2× RNA loading dye to 25 μL of sample. Prepare molecular weight marker solution by mixing 1 μL of ssRNA low-range ladder to 9 μL of nuclease-free water, followed by 10 μL of 2× RNA loading dye. Heat the samples at 92 °C for 4 min. In two wells load 20 μL of sample and in one well 10 μL. Load the marker in the last lane (see Note 6).

  3. Run the gel at 180 V until the lower (dark blue) dye is close to the bottom, approximately 70 min. Incubate the gel with 50 mL of TBE buffer containing 5 μL SYBR-Gold dye for 5 min. Wash the gel twice with nuclease-free water.

  4. Visualize and record the gel under UV light.

  5. Prepare 0.5 mL RNase-free non-stick tubes by piercing a hole in the bottom using an 18G syringe needle. Cut the gel on transilluminator as indicated in Fig. 3. Put gel pieces into 0.5 mL tubes and then place the tubes into a 1.5 mL collection tube. Centrifuge at 20,000 × g, for 3 min at 4 °C. Remove 0.5 mL tube and add 300 μL nuclease-free water.

  6. Shake samples on the thermomixer at 157 × g for 10 min at 68 °C, and then freeze them on dry ice for 10 min. Thaw samples at room temperature for 10 min and then incubate on thermomixer at 157 × g for 10 min at 68 °C.

  7. Cut the tips of the P1000 barrier tips, transfer the sample (with gel pieces) onto a Costar SpinX column, and spin at 20,000 × g for 3 min at 4 °C.

  8. Add to each tube 2 μL of GlycoBlue, 33 μL of 3 M sodium acetate pH 5.5, and 900 μL of 100% ethanol. Vortex to mix. Put at −20 °C overnight. Next morning precipitate RNA as described in Subheading 3.4.

3.6. RNA Fragmentation

Fragment RNAs longer than 300 nt to 50–200 nt long fragments using the following protocol:

  1. Resuspend RNA obtained in Subheading 3.5 in 11 μL of nuclease-free water. Transfer to PCR tubes and place in the thermocycler.

  2. Heat the samples for 2 min at 95 °C to denature RNA.

  3. Add 1 μL of 10× Fragmentation Reagent mix. If you are dealing with larger number of tubes, keep the tubes on ice. This will ensure that most of the RNA stays denatured. Keeping samples at the room temperature will allow for the slow refolding of RNA.

  4. Return the samples to thermomixer and incubate them for 2 min at 95 °C. Add 1 μL of stop solution and place the samples on ice.

  5. Purify the RNA on 10% TBE-urea gel. Extract 50–200 nt long fragments and precipitate RNA as described in Subheading 3.5 (see Note 7).

3.7. Library Preparation

3.7.1. RNA 3′ End Dephosphorylation

  1. Determine RNA concentration by NanoDrop. Low RNA concentration should be measured by Qubit or Bioanalyzer.

  2. Prepare 11 μL reaction mixture by mixing 7 μL of RNA sample with 1.1 μL of 10× PNK buffer, 1 μL of Superase-In, and 2 μL of T4 polynucleotide kinase. Incubate reaction mixture at 37 °C for 1 h, followed by 3 min at 90 °C to inactivate the enzyme.

3.7.2. Template-Switching Reaction Using TGIRT-III

  1. In a PCR tube prepare 17.5 μL reaction mixture by mixing nuclease-free water with 4 μL of 5× TGIRT reaction buffer, 2 μL of 50 mM DTT, ~50 ng of dephosphorylated RNA sample, and 2 μL 10× TGIRT-III RT/template-primer substrate mix (see Note 8).

  2. Preincubate reaction mixture at room temperature for 30 min, and then add 2.5 μL of 10 mM dNTPs.

  3. Incubate the reaction at 60 °C for 60 min (see Note 9).

  4. Add 1 μL of 5 M NaOH and incubate the sample at 65 °C for 15 min.

  5. Cool to room temperature and neutralize sample with 1 μL of 5 M HCl.

  6. To each sample add 100 μL of 10 mM Tris pH 8.0, 13 μL of 3 M sodium acetate pH 5.5, 3 μL of GlycoBlue, and 600 μL of 100% ethanol. Incubate overnight at −20 °C. Next morning precipitate cDNA as described in Subheading 3.4.

  7. Pre-run 8% TBE gel at 155 V for at least 20 min.

  8. Resuspend precipitated cDNA in 5 μL of 10 mM Tris pH 8.0 and add 1.25 μL of 5× DNA loading dye. Run gel at 155 V for 40–45 min.

  9. Size-select cDNA using 10 bp ladder as a guide. Add 300 μL of nuclease-free water to cut gel pieces, and extract cDNA from gel pieces by following the general protocol presented in Subheading 3.5. Precipitate cDNA overnight, at −20 °C, by adding 3 μL of GlycoBlue, 33 μL of sodium acetate pH 5.5, and 900 μL of 100% ethanol. Next morning precipitate cDNA as described in Subheading 3.4 (see Note 10).

3.7.3. Oligo Adenylation of Illumina Read 1 Sequencing Primer (R1R DNA)

  1. In a PCR tube prepare 20 μL reaction mixture by combining 2 μL of 10× 5′ DNA Adenylation Reaction Buffer, 2 μL of 1 mM ATP, 1 μL 100 μM R1R DNA, and 2 μL of Mth RNA Ligase. We usually set up 4–8 parallel reactions.

  2. Incubate reactions in a thermocycler at 65 °C for 1 h.

  3. Incubate samples at 85 °C for 5 min to inactivate the enzyme.

  4. Clean up the adenylated R1R DNA with an Oligo Clean & Concentrator kit and elute in 10 μL of nuclease-free water to obtain 10 μM adenylated R1R DNA. If doing several adenylation reactions in separate PCR tubes, combine them for a cleanup since higher elution volume helps with consistent and efficient recovery of adenylated oligos.

  5. Check the extent of adenylation by running the sample on a 20% TBE-7 M-urea gel.

3.7.4. Thermostable Ligation

  1. In a PCR tube prepare 20 μL reaction mixture by combining 2 μL of 10× NEBuffer 1, 2 μL of 50 mM MnCl2, 4 μL of 10 μM adenylated R1R DNA, 10 μL of cDNA from template-switching, and 2 μL of Thermostable 5′ AppDNA/RNA Ligase.

  2. Incubate reactions in thermocycler at 65 °C for 2 h.

  3. Incubate samples at 90 °C for 3 min to inactivate the enzyme.

  4. Clean up the ligated cDNA with a MiniElute PCR Purification Kit and elute in 23 μL of nuclease-free water.

3.7.5. PCR Amplification

  1. In an Eppendorf tube prepare a 53 μL reaction mixture by combining 29.5 μL of nuclease-free water, 10 μL of 5× Phusion HF buffer, 1 μL of 10 μM Illumina multiplex primer, 1 μL of 10 μM Illumina barcode primer, 10 μL of cDNA, 1 μL of 10 mM dNTPs, and 0.5 μL of Phusion High-Fidelity DNA Polymerase.

  2. Divide reaction mixture among three PCR tubes. Heat cDNA at 98 °C for 5 s, then amplify it for 15, 18, or 21 cycles of 98 °C for 5 s, 60 °C for 10 s, and 72 °C for 12 s.

  3. Mix 17 μL of PCR product with 4.25 μL of 5× DNA loading dye, load on an 8% TBE gel, and run the gel at 155 V for 45 min. Stain the gel with SYBR gold.

  4. Size-select amplified DNA using 10 bp ladder as a guide. Add 300 μL of nuclease-free water to cut gel pieces, and extract DNA from gel pieces by following the general protocol presented in Subheading 3.5. Precipitate DNA overnight, at −20 °C, by adding 3 μL of GlycoBlue, 33 μL of sodium acetate pH 5.5, and 900 μL of 100% ethanol. Next morning precipitate as described in Subheading 3.4. Resuspend each library in 10 μL of 10 mM Tris pH 8.

3.7.6. qPCR Quantification

  1. For the quantification of the libraries we use KAPA library quantification kit. If the kit is being used for the first time, add 1 mL of 10× Primer Premix to the bottle of 2× KAPA SYBRFAST qPCR Master Mix (5 mL) and mix by vortexing. Aliquot this solution and store at −20 °C.

  2. Determine the total number of reactions that will be performed. Usually we run six DNA standards in triplicate and each library dilution in duplicate. Using a NanoDrop, estimate the concentration of each library and determine which dilutions to prepare to stay within the dynamic range of the assay. A 1:5000 and 1:10,000 dilution usually fall around the midpoint of the assay standards.

  3. Prepare 1:5, 1:50, 1:5000, and 1:10,000 library dilutions in 10 mM Tris pH 8 buffer.

  4. For each reaction, prepare the following in a 96-well PCR plate: 6 μL of Master Mix containing primers, and 4 μL of either DNA standard or specific library dilution. Seal the plate with optical adhesive film.

  5. Run the plate with the following program in the qPCR machine: 95 °C for 5 min, and 35 cycles of 95 °C for 30 s, and 60 °C for 45 s.

  6. Use the KAPA analysis template to calculate slope and intercept of the standard curve, to convert the average Cq score for each library dilution to pM, to calculate the average size-adjusted concentration (in pM) for each dilution, and to calculate the size-adjusted concentration for the original undiluted library.

  7. Prepare 15 μL of 10 nM library solution, containing up to 20 libraries. Store individual libraries in RNase-free low-retention Eppendorf tubes at −20 °C (see Note 11).

  8. Check the quality of the library on Bioanalyzer prior to submitting sample for sequencing on an Illumina HiSeq4000 or similar (see Note 12).

3.8. Sequencing Read Mapping and Analysis

3.8.1. Sequence Processing and Alignment

  1. Prior to bioinformatic analysis it is important to de-multiplex sequences if multiple samples were run within one sequencing lane. De-multiplexing for our samples was performed by the Center for Advanced Technology at UCSF.

  2. Upload sequencing data to the Galaxy web platform and use the public server at usegalaxy.org to analyze the data [28 ] (see Note 13).

  3. Process reads with FASTQ Groomer [29] and then remove adapters using Clip tool also available through Galaxy web platform.

  4. Go to Ensembl Bacteria Genome Database (EMBL-EBI), https://bacteria.ensembl.org/index.html, and download E. coli BW25113 FASTA file and gtf file.

  5. Align sequences greater than 15 bp to the genome using Bowtie 2 with default options [30, 31]. Default settings for “sensitive-local” are the default option in “local-mode” (details are -D 15 -R 2 -N 0 -L 20 -i S,1,0.75) (see Note 14).

3.8.2. Enrichment Analysis of Reads

  1. Determine the raw counts per gene by using HTSeq-count script, which is available through Galaxy web platform [32]. Select intersection-nonempty mode to handle reads overlapping more than one feature. Summarize counts from regions A-D per replicate. Use this file to perform the enrichment analysis.

  2. For enrichment analysis of reads mapped to any set of genes use DESeq2 module. In DESeq2 specify the factor levels that will be analyzed (e.g., sample vs. control), and select all replicates belonging to a specific factor level. As an input data use summarized HTSeq-count data as described above. Use parametric fitting and leave on the following options: outliers replacement, outliers filtering, and independent filtering. For the control samples, we generated a library from the rRNA-depleted total RNA isolated from E. coli BW25113 strain (see Note 15).

3.8.3. Analysis of Stop Sites and Mismatches

  1. Download Integrated Genomic Viewer (IGV) [33]. Open E. coli BW25113 genome file. Open all BAM files and their corresponding BAM_index files.

  2. Select the gene of interest and determine the percent of mismatches for a specific nucleotide by cumulative analysis of all biological replicates (Fig. 4).

  3. To determine the 5′ end of the reads (stop sites) use script “make_wiggle” to convert sorted and indexed BAM files to wiggle files. This script was developed by the Weissman lab at UCSF and is readily available through Plastid [36]. The results can be readily visualized with IGV.

Fig. 4.

Fig. 4

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Examples of read profiles for specific RNAs displayed in Integrative Genomic Viewer. (a) Read profiles for tRNAGlnUUG displayed in Integrative Genomic Viewer (IGV) [33–35]. (b) Read profiles for tRNAHisGUG displayed in IGV. tRNAs were isolated after immunoprecipitation of FLAG-tagged C118A RlmN. The depth of the reads (counts) displayed at a specific locus is represented as a gray bar chart (top panel). Alignment of individual reads is represented in the bottom panel. Known modifications are represented using abbreviations and were taken from the MODOMICS database [35]. Abbreviations: 4-thiouridine (s4U), dihydrouridine (D), queuosine (Q), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), 2′-O-methyluridine (Um), 5-carboxymethylamino-methyl-2-selenouridine (cmnm5se2U), 5-methyluridine (m5U), 2-methyladenosine (m2A), and pseudouridine (Ψ)

Acknowledgments

This work was supported by UCSF Program for Breakthrough Biomedical Research (PBBR) Postdoctoral Grant (to V.S.), NIAID R01AI137270 (to D.G.F.), UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation (to D.E.W.), and NIH Director’s Early Independence Award DP5OD017895 (to D.E.W.).

4 Notes

1.

Optimal expression time for an enzyme should be determined empirically prior to proceeding to the next step.

2.

Resin cannot stay in glycine buffer for longer than 20 min.

3.

When dealing with a very small sample volume, combine all the samples from a single experiment into a single tube prior to leaving the sample on a rotator.

4.

After centrifugation remove the ethanol using P1000 pipette. Recap the tube, and pulse centrifuge to bring down the remaining ethanol. Remove the remaining liquid using P200 pipette. Leave the tube open at room temperature before proceeding to another sample. By the time all the samples are finished, the pellets should be sufficiently dry. Do not overdry the pellet since it can be hard to re-solubilize RNA.

5.

Perform Western blot analysis to ensure that the enzyme was successfully digested. We use monoclonal anti-FLAG M2-peroxidase (HRP) antibody.

6.

Prior to loading the samples flush the remaining urea out of the wells using P1000 pipette. This will decrease smearing and abnormal band shapes. Additionally, it is advisable to load a smaller amount of sample in one of the lanes to better see discrete bands, since loading a large amount of sample can lead to increased smearing in the gel.

7.

Under these conditions, the extent of fragmentation will depend on the initial amount of RNA. If substantial amount of RNA is not successfully fragmented, extract the RNA longer than 300 nt, and repeat the fragmentation step. Make sure to decrease the time for the re-fragmentation step (e.g., from 2 min to 1 min, or less).

8.

Add RNA sample and enzyme/template-primer mix last.

9.

For long or heavily modified RNAs, such as tRNAs, it is necessary to run the reaction for 60 min. For short RNAs 5–15 min is usually sufficient, but the exact time should be determined empirically.

10.

In case no pellet is observed after the first centrifugation step, add 1 μL of GlycoBlue, place sample on dry ice for at least 30 min, thaw sample at room temperature, and then repeat centrifugation step. Only then remove the supernatant and perform the wash step with cold 80% ethanol.

11.

When submitting multiple samples within one sequencing lane, approximately equal amounts of each library should be added.

12.

For our application and for cost-effectiveness, 50-nucleotide single-end runs are sufficient.

13.

When uploading large sequencing files, FileZilla, an open-source software, can be used.

14.

If applying this method to a different system, we suggest aligning sequences to the genome of interest using both Bowtie 1 and Bowtie 2 under various settings and then comparing results.

15.

DESeq2 considers the variability between the replicates and normalizes read counts to account for differences in sequencing depth between samples, reporting fold change values between the sample and the control. In our analysis, we use a fourfold increase in abundance and adjusted P value of <0.01 as our threshold for identifying substrates in samples where TGIRT was used as reverse transcriptase. DESeq2-adjusted P-values are adjusted for multiple-comparison testing and are used to lower the false-positive detection.

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