Comparative RNA Structure Analysis of Nascent and Mature Transcripts in Saccharomyces cerevisiae

Summary

RNA secondary structure has primarily been observed in mature RNA with structure probing methods. Co-transcriptional Structure Tracking sequencing (CoSTseq) unifies nuclear run-on, which has been used to study Polymerase position on nascent RNA, with structure probing. CoSTseq thereby enables observation of RNA secondary structure in RNA under active transcription.

Abstract

During transcription, nascent RNA starts base pairing as it exits RNA polymerases (Pols). This base pairing permits the formation of RNA structures that critically influence gene expression at the level of RNA processing, translation, and stability. Established methods to study RNA secondary structure are limited to mature transcripts, while little is known about folding states. Moreover, the relatively lower abundance (< 1%) and transient nature of nascent RNA complicate its isolation and characterization. Co-transcriptional Structure Tracking (CoSTseq) leverages transcriptional run-on with biotin-NTP and dimethyl sulfate (DMS) probing to simultaneously acquire Pol position and base pairing status of nascent transcripts. In Saccharomyces cerevisiae, CoSTseq yields the sequence and structural information near the 3'-end of nascent RNAs transcribed by any of the three RNA Pols. During transcriptional run-on, the biotin-NTP incorporated at the active site effectively stalls Pols. Then, treatment with DMS methylates unpaired A, C, and U nucleotides. Subsequent biotin enrichment and cDNA synthesis with a template-switching reverse transcriptase enables paired-end sequencing and the computation of DMS reactivities as a function of Pol position. CoSTseq is readily performed side-by-side with DMS probing (DMS-MaPseq), enabling capture of the folded mature transcript as well. Here, a detailed protocol is presented for parallel CoSTseq and DMS-MaPseq, including transcriptional run-on, library preparation, and data analysis.

Introduction

RNA can fold into secondary and tertiary structures due to base pairing within RNA molecules, and these structures can further be influenced by proteins that act as chaperones to guide RNA folding¹. RNA structure can be highly dynamic, where cellular RNAs can conform to a range of structures defined by the thermodynamic landscape to generate ensembles of possible RNA conformations². Dynamic conformational changes have the potential to affect gene regulation and expression^3,4. Conversely, RNA can also adopt highly favored structures that are tightly related to its....

Protocol

NOTE: Before beginning, make sure all the buffers listed in Table 1 are prepared. All the reagents should be prepared in nuclease-free water. Filter sterilization of buffers is recommended when using non-molecular biology grade chemicals/reagents for buffer preparation. For sections 1 through 4, prepare the following in advance:

1. Preparation of materials and reagents

1. Prepare a 10% sarkosyl (v/v) solution at least 1 day in advance to allow sufficient time for complete dissolution. Filter-sterilize the homogeneous solution with a 0.22 µm filter. On the day of the experiment, use the 10% sarkosyl....

Results

This section presents the actual results generated by implementing the CoSTseq workflow and analysis, as described in this protocol. Firstly, this section describes expected outcomes following quality assessment of successful library preparations before and after sequencing. Prior to sequencing, researchers can use a test PCR and TapeStation analysis to confirm the presence of RNA following biotin enrichment. This indicates successful isolation of nascent RNA during CoSTseq library preparation. Data quality assessment af.......

Discussion

RNA starts to fold co-transcriptionally due to faster kinetics of base-pairing compared to the rate of synthesis^24,25,26,27. Our current knowledge of nascent RNA folding comes from single-molecule studies of prokaryotic RNAs, in vitro probing, or in silico approaches. In this protocol, a detailed workflow of CoSTseq is presented; this technique allows in vivo detectio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10mM ATP	Invitrogen	18330019
10mM Biotin-11-CTP	Jena biosciences	NU-831-BIOX
10mM GTP	Invitrogen	18332015
10mM UTP	Invitrogen	18333013
10X NEBuffer 1	New England Biolabs	B7001S	Used in step 7.2.1
10X Phosphate Buffered Saline (PBS)	Gibco	70011-044
20% SDS	RPI	L23100-500
Acid phenol:chloroform	Ambion	AM9722
AMPure XP beads for size selection	Beckman Coulter	A63880	Used for Size Selection in step 7.5.1
Bacto Peptone	Gibco	211677
Bacto Yeast Extract	Gibco	212750
Bicine	Sigma Aldrich	B3876-100G
Chloroform	Sigma Aldrich	319988
D-(+)-GLUCOSE	Sigma Aldrich	G5767-500G
DIFCO AGAR	BD DIFCO	214010
Dimethyl Sulfate	Sigma Aldrich	D186309-5ML
Dynabeads™ MyOne Streptavidin C1 beads	Invitrogen	65001	Used for Biotin Pulldown in Section 4.1
Dynabeads™ mRNA DIRECT™ Purification Kit	Invitrogen	61011	Used for Poly A selection in Section 5.1
EDTA, pH 8, 0.5M	Sigma Aldrich	03690-100ML
Ethanol	Sigma Aldrich	E7023-500ML
GlycoBlue	Invitrogen	AM9516	Co-precipitant used in step 4.2.7 and 5.2.4
Induro® Reverse Transcriptase	New England Biolabs	M0681S	RT enzyme used in step 6.3.1
Isoamyl alcohol	Sigma Aldrich	W205710-1KG-K
Isopropanol	JT-BAKER	9084-05-01
KAPA HiFi HotStart PCR Kit	Roche	07958889001	High-fidelity DNA Pol used in 7.3.2 and 7.4.1 for PCR reactions
Magnesium Chloride	Sigma Aldrich	SLCM2154
MinElute PCR Purification Kit	Qiagen	28004	Used for DNA clean up in step 6.3.3 and 7.2.2
Mth RNA Ligase	New England Biolabs	M2611A
Oligo Clean & Concentrator	Zymo Research	D4060	Suggested for DNA Oligo clean up in step 7.1.2
Potassium Acetate	Sigma Aldrich	236497-500G
Potassium Chloride	JT Baker	3040-01
Potassium Hydroxide	Avantor	6984-04-01
RNA Clean & Concentrator-5	Zymo Research	R1014	RNA clean up kit used in step 5.3.2 after PNK treatment
RNaseOUT™ Recombinant Ribonuclease Inhibitor	Thermo Fisher Scientific	10777019	RNase inhibitor used in step 5.3.1 during PNK treatment
Sarkosyl	IBI Scientific	IB07080
Sodium Acetate	Quality Biological	351-035-721
Sodium Chloride	Sigma Aldrich	S5150-1L
Sodium Hydroxide	Macron	7708-10
SUPERase·In™ RNase Inhibitor (20 U/μL)	Thermo Fisher Scientific	AM2696	RNase inhibitor used in step 6.3.1
T4 Polynucleotide Kinase	New England Biolabs	M0201S
Thermostable 5’ App DNA/RNA Ligase	New England Biolabs	M0319L
Tris-HCl, pH 7.4, 1M	Thermo Scientific	J60202.K2
Triton X-100	TEKNOVA	T1105
TRIzol™ Reagent	Invitrogen	15596-026	For nascent RNA elution in Section 4.2; also referred as "RNA reagent" in Section 4
β-mercaptoethanol	Sigma Aldrich	M6250-1L