哺乳动物新生 RNA 转录本 5′-单磷酸化末端的全基因组定位

Michael A. Cortázar; Nova  Fong; David L. Bentley

doi:10.21769/BioProtoc.4828

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Peer-reviewed

Genome-wide Mapping of 5′-monophosphorylated Ends of Mammalian Nascent RNA Transcripts

哺乳动物新生 RNA 转录本 5′-单磷酸化末端的全基因组定位

Michael A. Cortázar email

NF Nova Fong

DB David L. Bentley

发布: 2023年09月20日第13卷第18期 DOI: 10.21769/BioProtoc.4828 浏览次数: 1905

评审: Gal HaimovichMarion HoggRohini Ravindran Nair

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Abstract

In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5′-monophosphorylated (5′-PO₄) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5′-PO₄ ends can be a target of further processing or be subjected to 5′-3′ exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5′-PO₄ ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5′-PO₄ RNA ends with the 3′-hydroxyl group of a 5′ adaptor (5′-PO₄ Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5′-PO₄ groups precisely. This protocol successfully detects the 5′monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5′-3′ exonuclease Xrn2, 5′-PO₄ Bru-Seq maps the 5′ splice sites of debranched introns and mRNA and tRNA 3′ end processing sites cleaved by CPSF73 and RNaseZ, respectively.

Key features

• Metabolic labeling for brief periods with bromouridine focuses the analysis of 5′-PO₄ RNA ends on the population of nascent transcripts that are actively transcribed.

• Detects 5′-PO₄ RNA ends on nascent transcripts produced by all RNA polymerases.

• Detects 5′-PO₄ RNA ends at single nucleotide resolution.

Keywords: 5′-PO4 Bru-Seq (5'-PO4 Bru-Seq)

5′-monophosphorylated ends (5'-单磷酸化末端)

Nascent transcripts (新生转录物)

RNA (RNA)

Bromouridine labeling (溴尿苷标记)

Immunoprecipitation (免疫沉淀)

Background

Tracking genome-wide active transcription and its regulation has been made possible by several complementary approaches (Wissink et al., 2019). These include isolation of chromatin-associated RNAs (Bhatt et al., 2012, Mayer et al., 2015, Weber et al., 2014), RNA polymerase–associated RNAs (Churchman and Weissman, 2011, Nojima et al., 2015, Fong et al., 2017), or nascent transcripts pulse-labeled with nucleoside analogs such as 4-thiouridine (Schwalb et al., 2016, Kenzelmann et al., 2007, Rabani et al., 2011, Herzog et al., 2017, Muhar et al., 2018, Schofield et al., 2018), 5-ethenyluridine (Jao and Salic, 2008), or bromouridine (Paulsen et al., 2014).

In eukaryotic cells, endonucleolytic cleavage of the nascent transcript is used to release fully transcribed RNAs from chromatin or to release small RNAs from longer precursors during transcription [i.e., micro RNAs (miRNAs) and intron-encoded small nucleolar RNAs (snoRNAs)]. Most of these cleavage events are carried out by nucleases that leave 5′-phosphate (5′-PO₄) and 3′ OH ends, including CPSF73, Int11, RNaseP, RNaseZ, and Drosha.

Recently, POINT-5 technology identified 5′ ends generated at cleavage sites of RNA pol II–associated transcripts from runoff products of 5′ RACE in reverse transcription reactions (Sousa-Luis et al., 2021), but this approach does not inform on the identity of the chemical group at these 5′ RNA ends. Previously, methods have been developed to specifically map 5′-PO₄ RNA ends by virtue of their ability to be ligated to adaptors by RNA ligase I (Harigaya and Parker, 2012, German et al., 2008).

Here, we describe 5′-PO₄ Bru-Seq for direct detection of 5′-PO₄ groups in nascent transcripts produced by all RNA polymerases in the cell (Figure 1). The method was previously validated and used to identify targets of the Xrn2 exonuclease (Cortazar et al., 2022). In this method, total RNA is fragmented with Micrococcal Nuclease (MNase) that produces 5′ OH and 3′ PO₄ ends, and nascent transcripts are enriched by immunoprecipitation of 5-bromouridine pulse-labeled molecules, followed by repair of 3′ phosphates with T4 polynucleotide kinase, ligation of 5′ and 3′ adaptors, and PCR amplification of sequencing libraries. Only RNA fragments with native 5′-PO₄ ends can be incorporated into these libraries (Harigaya and Parker, 2012). Unique molecular identifiers (UMIs) are included to allow removal of PCR duplicates. The molecular events that result in 5′-PO₄ groups on nascent RNA ends detected by this method potentially include cleavage by endonucleases, intron debranching, and decapping.

Figure 1. The 5′-PO₄ Bru-Seq protocol. 5′-PO₄ Bru-Seq enriches transcripts being extended by actively transcribing polymerases via metabolic labeling with bromouridine, fragmentation by micrococcal nuclease (MNase), and immunoprecipitation. The 3′-ends generated by MNase cleavage events are repaired by T4 polynucleotide kinase (PNK) in the absence of ATP. 5′-PO₄ ends of nascent transcripts are then ligated to the 3′-hydroxyl group of a 5′ adaptor, amplified by PCR, and deep sequenced using Illumina sequencing technology.

Materials and reagents

Mammalian cell type of interest (e.g., HEK293, ATCC, catalog number: CRL-1573)
Cell culture media supplemented with 10% serum (e.g., DMEM supplemented with 10% FBS; DMEM, Thermo Fisher Scientific, catalog number: 11995040; FBS, VWR, catalog number: 97068-085)
10 cm tissue culture dishes (e.g., Genesee Scientific, catalog number: 25-200)
5/10 mL sterile serological pipets (e.g., Genesee Scientific, catalog numbers: 12-102, 12-104)
15/50 mL conical bottom centrifuge tubes (e.g., Corning, catalog numbers: 05-538-59A, 05-526B)
1.7 mL microcentrifuge tubes (e.g., Genesee Scientific, catalog number: 22-282)
TRIzol reagent (Thermo Fisher Scientific, catalog number: 15596018)
Chloroform (Fisher Scientific, catalog number: BP1145-1)
Isopropanol (Fisher Scientific, catalog number: BP2618-1)
80% ethanol, molecular biology grade (e.g., Thermo Fisher Scientific, catalog number: T08204K7)
GeneRuler 1 kb Plus DNA ladder (Thermo Fisher Scientific, catalog number: FERSM1333)
5 M NaCl, RNase-free (Thermo Fisher Scientific, catalog number: AM9760G)
Micrococcal nuclease (MNase) (2,000,000 gel units/mL) (New England Biolabs, catalog number: M0247S)
10× MNase reaction buffer (New England Biolabs, catalog number: B0247S)
0.5 M EGTA, pH 8.0, DNase and RNase Free (Thermo Fisher Scientific, catalog number: NC1874048)
Nuclease-free water (Thermo Fisher Scientific, catalog number: AM9939)
10× phosphate-buffered saline (PBS), pH 7.4, RNase-free (Thermo Fisher Scientific, catalog number: AM9624)
5-bromouridine (Sigma-Aldrich, catalog number: 850187)
Triton X-100 (Sigma-Aldrich, catalog number: X100)
1 M DTT (Thermo Fisher Scientific, Catalog number: P2325)
Superase-In^TM RNase inhibitor (Invitrogen, AM2696)
Protein G magnetic beads (Pierce Thermo Scientific, catalog number: 88848)
Purified mouse anti-BrdU antibody (clone 3D4, RUO) (BD Pharmingen, catalog number: 555627)
Qubit^TM RNA High Sensitivity Assay kit (Thermo Fisher Scientific, catalog number: Q32852)
Invitrogen^TM Qubit^TM assay tubes (Thermo Fisher Scientific, catalog number: Q32856)
T4 polynucleotide kinase (Thermo Fisher Scientific, catalog number: EK0031)
10× reaction buffer A (Thermo Fisher Scientific, catalog number: EK0031)
RNA Clean & Concentrator^TM-5 kit (ZYMO RESEARCH, catalog number: R1013, R1014)
QIAseq miRNA library kit (Qiagen, catalog number: 331502)
QIAseq miRNA Index kit IL UDI (e.g., UDI-B-96, Qiagen, catalog number: 331625)
10× TBE Buffer (Thermo Fisher Scientific, catalog number: AM9863)
1:20 dilution of MNase (100,000 gel units/mL) (see Recipes)
50 mM 5-bromouridine (see Recipes)
IP buffer (see Recipes)

Software

Removal of UMI duplicates: UMI-tools (Smith et al., 2017)
Removal of adaptor sequences: Cutadapt (Martin, 2011)
Genomic alignment of sequencing reads: Bowtie2 (Langmead and Salzberg, 2012)
BAM file processing: Samtools (Li et al., 2009)
Integrative Genomics Viewer (IGV) (Robinson et al., 2011)
Conversion of BAM files to BED files: bedtools (Quinlan and Hall, 2010)
Conversion of bedGraph files to BigWig: bedGraphToBigWig (Kent et al., 2010)

Equipment

1.5 mL microcentrifuge magnet stand (e.g., Thermo Fisher Scientific, catalog number: 12321D)
Centrifuge 5430 R (e.g., Eppendorf, catalog number: 022620601)
Thermal cycler (e.g., Bio-Rad, model: C1000 Touch, catalog number: 1851148)
Qubit fluorometer (Thermo Fisher Scientific, catalog number: Q33238)

Procedure

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如何引用

Cortázar, M. A., Fong, N. and Bentley, D. L. (2023). Genome-wide Mapping of 5′-monophosphorylated Ends of Mammalian Nascent RNA Transcripts. Bio-protocol 13(18): e4828. DOI: 10.21769/BioProtoc.4828.