Segmental Labeling of RNA with Stable Isotopes

Duss et al. in 2010  reported the development of a method using ¹³C and ¹⁵N labeled nucleoside triphosphates (NTPs) for the segmental labeling of RNA with stable isotopes. Recent advancements made in mass spectrometry, in bioinformatics as well as in the development of enrichment methods for stabil isotopes, the use of stabile isotope in biochemistry, biology, biotechnology, molecular biology, and medical research has now become common.

According to Duss et al. enzymatic ligation of shorter modified synthetic RNA segments with longer fragments produced by in vitro transcription is expected to become the method of choice for studying biological important RNAs. Segmental labeling of RNA with stable isotopes and the use of ligation methods to incorporate synthetic RNA pieces containing modified nucleotides into RNA promises to overcome present limitations in obtaining structural information on RNA. Several researchers are now tackling this problem. The structural model of the non-coding RNA RsmZ protein sponge was reported by Duss et al. using this approach to allow for the elucidation of the solution structure via a combination of nuclear magnetic resonance and electron paramagnetic resonance spectroscopy.   

Figure 1: Non-coding RNA RsmZ acts as a protein sponge. The bacterial Csr/Rsm system is considered to be the most general global post-transcriptional regulatory system responsible for bacterial virulence. NcRNAs such as CsrB or RsmZ activate translation initiation by sequestering homodimeric CsrA-type proteins from the ribosome-binding site of a subset of messenger RNAs. The structural model of this complex was reported by Duss et al. in 2014.

Natural and synthetic RNA molecules are frequently labeled using radioisotopes or fluorophores. Unfortunately, both labeling methods are unsuitable for clinical studies. The reason for this is that both labeling methods have side effects such as the risk of radiation exposure or the different metabolic behavior of the fluorophore-conjugate in comparison to the unconjugated active compound studied. RNA is now widely recognized for its function in regulating gene expression. Many RNA molecules including riboswitches, miRNAs, and large non-coding RNAs are now thought to play roles in gene regulation. Only approximately 1.5% of the human genome encodes proteins while 60 to 70% of it is transcribed into RNA. However, only circa 2% of structures deposited in the Protein Data Bank account for RNA.

Nuclear magnetic resonance (NMR) is now considered to be the method of choice for solving RNA structures.  Unfortunately, large RNA structures are hard to analyze due to spectral overlap observed in NMR spectra of RNA. Segmental labeling of RNA is therefore considered to be needed for the study of RNAs by NMR in combination with measurements of residual dipolar couplings (RDC) and paramagnetic relaxation enhancement (PRE). Also, site-specific incorporation of isotopes can also be useful for solving phases in X-ray crystallography.

The use of isotope-labeled nucleotide triphosphates (NTPs) for the generation of multiple segmentally labeled RNAs is very useful for structural studies to expand our knowledge in the structural biology of RNA. The research group presented a method that is considered to be a fast, efficient and sequence-independent method for segmental labeling of RNA. The method was tested using the 72 nt RsmZ RNA product that was isotopically segmentally labeled for structural investigations by NMR. The method is based on a combination of co-transcriptional ribozyme cleavage, sequence-specific RNase H cleavage, and cross-religation using either T4 RNA or T4 DNA ligase. PrrB/RsmZ RNA are part of a group of non-coding RNAs (ncRNAs) found in bacteria. Research using Legionella pneumophila  indicates that the ncRNAs RsmY and RsmZ together with the proteins LetA and CsrA are part of a regulatory cascade and appear to be regulated by RpoS sigma-factor.

Outline of the Method

This approach is based on the transcription of two full-length RNAs. These RNAs have identical sequence but one is unlabeled whereas the other is isotopically labeled. The transcribed RNAs are flanked at the 5’-end by a hammerhead (HH) ribozyme in cis and at the 3’-end by a minimal sequence required by the Neurospora Varkud satellits (VS) ribozyme for cleavage in trans.

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Step 1: Co-transcriptional ribozyme cleavage. Transcribed RNAs are flanked at the 5’-end by a hammerhead (HH) ribozyme in cis and at the 3’-end by the minimal sequence required by the Neurospora Varkud satellite (VS) ribozyme for cleavage in trans. The two ribozymes (in cis or in trans) cleave co-transcriptionally. The result is two homogenous termini, a 5’-hydroxyl and 2’/3’-cyclic phosphate for the full-length RNA, which are purified before the next step.

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Step 2: Site-specific RNase H cleavage. After purification, the two transcribed RNAs are site-specifically cleaved by RNase H using a guide 2’-O-methyl-RNA/ DNA splint. This reaction yields an acceptor fragment (5’-fragment) with two hydroxyl termini and a donor fragment (3’-fragment) with a phosphate at its 5’-end and a cyclic 2’/3’-phosphate at its 3’-end.

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Step 3:  Cross-religation between the labeled and unlabeled fragment. After separation of the two fragments from each cleavage reaction, T4 RNA or DNA ligase is used for re-ligation. This reaction results in two segmental isotope labeled RNA fragments. Either the 5’-fragment or the 3’-fragment is labeled with the isotopes.    

For the method to work well a fast and efficient denaturing anion-exchange HPLC purification step is needed followed by n-butanol extraction or dialysis to get rid of urea and salts.

Duss et al. estimated that for a two-piece ligation, 5 to 7 days are required in total. A total of 2 to 3 days are needed for Step 1 (1 day transcription optimization, 1 to 2 days large-scale transcription and purification), 1.5 to 2 days for Step 2 (0. 5 to 1 day RNase H cleavage optimization, 1 day large-scale cleavage and purification) and 1.5 to 2 days for Step 3 (0.5 to 1 day ligation optimization, 1 day large-scale ligation and purification).

Methods used for this approach 

• Vector construction and plasmid purification,

• RsmE protein expression and purification

• RNA purification,

• RNA transcription and co-translational ribozyme cleavage (IVT),

• Segmental isotope labeling,

• Sequence-specific RNase H cleavage,

• RNA ligation with T4 RNA and DNA ligase, and

• NMR spectrocopy,

• EPR spectroscopy, 
  

• DEER experiment (Double Electron-Electron Resonance, also known as PELDOR or Pulsed ELDOR),

• Electrophoretic mobility shift assaya (EMSA),

• Isothermal titration calorimetry (ITC) binding experiments.
  

Isotope labeling

Labeled nucleoside triphosphate (NTPs) can be either purchased from a provider such as Cambridge Isotope Laboratories (CIL) or prepared from ¹³C, 15N-labeled E. coli cultures. The labeled E. coli cultures are precipitated with sodium acetate and isopropanol, and hydrolyzed with S1 nuclease. The use of a boronate affinity gel column allows for the separation nucleoside monophosphates (NMPs). NMPs are converted to NTPs by an enzymatic phosphorylation step. Boronate affinity chromatographyis also used for the desalting of labeled NTPs.

Reference

Duss, O., Michel, E., Yulikov, M., Schubert, M, Jeschke, G., Allain, F.; Structural basis of the non-coding RNA RsmZ acting as a protein sponge. Nature 2014, 509, 588-592.

Olivier Duss, Christophe Maris, Christine von Schroetter and Fre´de´ric H.-T. Allain; A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage. Nucleic Acids Research, 2010, Vol. 38, No. 20 e188, doi:10.1093/nar/gkq756.

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