Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Summary

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Abstract

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.

In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Introduction

Primer extension¹ is a molecular method to determine the 5’ ends of specific RNA molecules up to a one base resolution. The advantage to other methods such as 5’-RACE (rapid amplification of cDNA ends) is the fast turnaround time and the ability to easily analyze a mixture of different lengths of RNA molecules.

This method works by subjecting RNA molecules to reverse transcription reactions using specific fluorescent primers, generating cDNA fragments of certain lengths. These cDNA molecules are run alongside traditional Sanger sequencing reactions² on denaturing polyacrylamide gels and can be detected by their fluorescence due to the use of fluorescently labeled primers. The lengths of the cDNA fragments are then assessed by comparison to the sequencing ladder, allowing the mapping of the 5’ RNA ends.

Traditionally, primer extension reactions are used in conjunction with radioactive isotopes to detect cDNA molecules on X-ray films. Due to health hazards, waste disposal issues and ease of handling, newer protocols utilize fluorescence for the detection of the primer extension with automated sequencers, albeit their sensitivity is slightly lower. Using fluorescently labeled primers, the recurring radio-labeling procedure can be omitted, as fluorescent primers are stable for a long time (more than a year in our hands).

The method we describe here utilizes an automated gel sequencer, but with slight modifications, capillary sequencers can be also used for the cDNA separation and detection³. The parallel nature of gel analysis makes it possible to detect even a small amount of RNA cleavage or processing. Another advantage is the high resolution of this method, as terminal cleavage or processing of even one base can be detected.

In regard to the detection of RNA cleavage or processing, typically two different types of primer extensions are distinguished. In one case, the enzymatic treatment is done in vitro using purified RNA and purified enzyme, whereas in the other case, the processing is done in vivo and the resulting RNA is purified. In both cases the RNA is subjected to a primer extension carried out in vitro, however, depending on the source of RNA, the method is either called an in vitro or in vivo primer extension. In the protocol we present here, we focus solely on the in vivo primer extension, because of ease of use (no purified proteins necessary) and the possibility to determine transcriptional starting points and processing at the same time. However, in vitro primer extensions are in principle set up the same way and this protocol can serve as a starting point.

The method illustrated here can be applied to many bacterial species as long as they are amenable to high purity and high-yield preparation of nucleic acids.

The research in our lab focuses on the regulatory scope of toxin-antitoxin (TA-) systems^4,5, a field in which the primer extension method is extensively used. TA-systems are small genetic elements present in prokaryotic genomes that consist of a stable and endogenously active toxic protein and a mostly unstable protein or RNA antitoxin that counteracts toxicity^6,7. Toxin activity is sometimes exerted by inhibition of replication, cell wall synthesis or other mechanisms, but most often by RNase activity^8,9. Typically, RNase specificity is determined by conducting different tests, one of which is the primer extension method. Primer extension reactions are well suited for this application, as a mixture of cleaved and full length fragments can be simultaneously analyzed to determine their 5’ ends. Using a mix of in vitro and in vivo primer extensions, the specific toxin RNase cleavage, e.g., sequence specificity can be determined^10-13.

Figure 1. Overview of primer extension procedure. Bacterial cultures are incubated and treated according to the experimental needs. Total RNA is extracted from the cells, treated with DNase I to remove DNA traces and subjected to a reverse transcription reaction using target specific fluorescent DNA primers yielding cDNA. Genomic DNA or plasmids are extracted and subsequently used for fluorescent Sanger sequencing reactions for size comparison with the cDNA fragments. Primer extension products are run alongside Sanger sequencing products on a denaturing urea polyacrylamide gel and analyzed with an automated laser and microscope. The sequencing base that lines up with the cDNA band is the last base of the 5’ cDNA end (blue arrow). More information in Fekete, et al. ³ Please click here to view a larger version of this figure.

An overview of the whole primer extension procedure can be found in Figure 1. Briefly, bacterial cells are cultured, harvested, the cell pellet lysed and the RNA extracted. Purified RNA is then treated with DNase I to remove traces of DNA molecules which could act as templates for the reverse transcriptase. Specific fluorescent primers are added to the RNA, hybridized to the region of interest and subsequently reverse transcribed, resulting in single stranded complementary DNA (cDNA). A sequencing ladder is created by traditional Sanger sequencing employing fluorescent primers and separated on a denaturing polyacrylamide gel alongside of the primer extension cDNA fragments. The resulting gel is analyzed by comparing the fluorescent bands, allowing the identification of the 5’ ends of interest. Transcriptional starting points and processing sites are then assessed individually by sequence comparisons.

Protocol

1. High Yield RNA Preparation RNA Isolation NOTE: High concentrations of total RNA are needed for the primer extension reaction. Spin column kits usually do not yield the amount of RNA needed (~5 – 16 µg in 5 µl volume). Therefore purification using the acid guanidinium thiocyanate-phenol-chloroform extraction method is recommended, outlined below. NOTE: Phenol is carcinogenic, toxic and corrosive. Please read the material safety data sheets and use under a fume hood with appropriate p…

Representative Results

As depicted in Figure 6, a primer extension reaction can be used to determine the transcriptional starting points of transcripts of interest and can help to deduce promoter regions (typically identified by -10 and -35 elements). The topmost (longest) cDNA fragment represents the 5’ end of the mRNA and thus can be easily mapped when compared to the sequencing ladder. Figure 6. Represen…

Discussion

Fluorescent primer extension is a simple and rapid method for determining the 5’ ends of RNAs, either for TSP- or secondary RNA processing identification. Due to the use of fluorescent primers, the reactions can be set up and run without additional security precautions (unlike in case of radioactively labeled primers). As the samples are detected by fluorescence, they can be imaged while the electrophoresis is in progress which allows rapid analysis in comparison to radioactive methods where X-ray films are commonl…

Materials

Name	Supplier	Catalog Number(s)	Comment
AMV Reverse Transcriptase (20-25 U/µl)	NEB / Roche	NEB: M0277-T / Roche: 10109118001
DNase I (RNase free)	Ambion (life technologies)	AM2222
FastPrep-24 Instrument	MPBio	116004500
Fluorescently labeled primers	Biomers	n/a	5’ DY-681 modification of “ordinary” DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.
Li-Cor 4200 Sequencer incl. ImagIR Data collection software	Li-Cor		Product discontinued
NanoDrop 2000	Thermo Scientific
Nuclease free water	Ambion (life technologies)	AM9915G
Plasmid mini preparation kit	QIAGEN	12125
RapidGel-XL-40% Concentrate	USB	US75863
RNA STORAGE BUFFER	Ambion (life technologies)	AM7000
Roti-Aqua-P/C/I	Carl Roth	X985.3	Alternative: “Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)” from Ambion (AM9720)
SUPERase•In RNase Inhibitor	Ambion (life technologies)	AM2696
Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP	GE Healthcare	RPN2538
TRIzol reagent	life technologies	15596-026
Zirconia/Silica Beads 0.1 mm	BioSpec	11079101z