Chapter 2 : RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification

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This chapter reviews the methods that have been successfully used to date for the identification of RNA modification and editing enzymes. The development of recombinant DNA and RNA techniques and techniques for chemical synthesis of DNA and RNA offers adequate alternatives for obtaining synthetic or semisynthetic unmodified or partially modified RNA substrates suitable for analyzing enzymatic posttranscriptional RNA modifications both in vivo and in vitro. Stepwise automated chemical synthesis of DNA oligonucleotides is widely used for producing starting material for gene construction, primers for PCR, DNA splints, or DNA probes for hybridization and deoxyribozymes. Two distinct approaches are generally used to gain the information on the precise location of modification or editing sites in RNA molecules. The first is based on direct sequencing of pure RNA and is applied mostly for the analysis of rather short RNA molecules, while the other is based on the use of reverse transcription with unfractionated RNA mixtures and specific oligonucleotide primers. Microinjection of antisense RNA, with the aim of blocking translation of selected mRNAs in the oocyte or fertilized frog eggs, revealed the existence of what was first identified as an unwinding RNA protein. With the recent access to complete genome sequences, it has become possible to search for homologous genes in the different genomes, to clone them into suitable vectors after amplification by PCR, and to express them in appropriate host cells.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2

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Image of Figure 1
Figure 1

Schematic procedures for construction of DNA templates that can be used for in vitro transcription into RNA using specific DNA-dependant RNA polymerases. On the left side, the starting material corresponds to pure chemically synthesized oligodeoxynucleotides, while on the right, the starting material corresponds to natural RNA genes present in isolated genomic DNA. The different steps of the procedure are indicated in boxes. DNA is represented by bars. A thick bar stands for the DNA portion corresponding to a promoter (P) of bacteriophage T7 or SP6 RNA polymerase. The asterisk stands for the restriction site needed for the linearization of the plasmid, before the transcription. The resulting “runoff” RNA transcript is indicated by a curved gray line. It can be radiolabeled internally on one of the four nucleotides with either P,H, or any other isotope by adding the corresponding radiolabeled nucleotide triphosphate into the transcription mixture. Hydroxyl and phosphate terminal groups are indicated only when these are essential. Intermediate purification steps by electrophoresis on agarose or polyacrylamide gels are not indicated.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 2

Site-specific cleavage of RNA. RNA fragments are prepared from long natural RNA by hybridization selection and addressed cleavages by RNase T1 (G specific). Short synthetic DNA, long complementary DNA (cDNA) obtained by reverse transcription of a natural RNA, or single-stranded phage DNA (ssDNA) may be used to protect the desired region of the targeted RNA from nuclease digestion. Modified nucleotides present in the natural RNA are illustrated by a black dot and a black square. If present in the RNA portion to be protected, they should allow formation of a perfect Watson-Crick base pair. “Mild conditions” means that a low ratio of RNase T1 to the amount of substrate to be digested is used; also, the digestion time is carefully controlled. After removal of the RNase T1 by phenolization, the resulting RNA fragments are purified by gel electrophoresis or any other purification procedure. The purified fragments have a 5′-hydroxyl group and a 3′-phosphate, which are not suitable for subsequent RNA ligation (see Fig. 5 and 6 ).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 3

Isolation of large RNA fragments. RNA is cleaved within an RNα-DNA hybrid by RNase Η or catalytic DNA enzyme. With complementary DNA 6 to 20 nucleotides long, multiple cuts (shown by arrows) arise in the hybrid region (left part of the figure). A site-specific single cut in RNA can be produced only when a short cDNA (4-mers) is flanked by two complementary 2'-O-methylated RNAs of 6 to 10 nucleotides on each side (central part of the figure). The DNA enzyme (DNAzyme) shown in the right part of the figure corresponds to a catalytic domain of 15 synthetic deoxynucleotides, flanked by two RNA-complementary domains of 7 to 8 deoxynucleotides on each side. The DNAzyme binds to the target RNA through Watson-Crick base pairs. The substrate is cleaved at a phosphodiester bond located between an unpaired purine and a paired pyrimidine residue. If modified nucleotides (illustrated by a black dot and a black square) are present in the target RNA, they should allow formation of a perfect Watson-Crick base pair in the RNA/DNA hybrid.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 4

Simple one-step procedure for isolating of RNA or RNA fragments from bulk RNA by the affinity purification technique. A DNA (synthetic or natural, represented by a thick bar) bearing a tail complementary to the target RNA is covalently linked via an appropriate spacer (represented by a zigzag line) to a solid support (usually agarose beads, paramagnetic beads or diol-silica beads). Alternatively, synthetic DNA biotinylated at its 5′ end is immobilized on avidin-coated agarose beads (which could also be paramagnetic). Bulk cellular RNA or fragmented RNA bearing the complementary region of the DNA tail is hybridized to the bound DNA. The beads are then collected by centrifugation or with a magnet and washed several times. The trapped RNA is recovered by using appropriate experimental conditions (use of low salt concentration, high temperature, proteinase K). After concentration, the isolated RNA is further purified by gel electrophoresis or by any other technique such as anion exchange or adsorption chromatography.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 5

In vitro recombination of RNA fragments (natural or synthetic). The joining reactions can be catalyzed either by T4 DNA ligase or by T4 RNA ligase. With DNA ligase (A), the RNA acceptor and the RNA donor have to be bridged with a DNA splint (black thick line) complementary to the two RNA extremities, thus forming a sort of nicked duplex. With RNA ligase (B), the RNA donor and the RNA acceptor have to be self-complementary in the vicinity of the ligation sites, thus forming a stem-loop structure. RNA recombination can also occur with RNA partners that cannot form such stem-loop structures, but the yield of the ligation reaction is usually dramatically low. For both ligation reactions, the 3′ end of the acceptor RNA should have free 2'- and 3′-hydroxyl groups, while the 5′ end of the donor RNA molecule has to be phosphorylated. If this 5′-phosphate is radiolabeled with P, the resulting recombinant RNA will contain a site-specific radiolabeled phosphate precisely at the ligation site. The black dot stands for naturally occurring modified or edited nucleotides.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 6

Strategies for site-specific replacement of a nucleotide, Ζ (modified or not), by another nucleotide, Y (modified or not), in a naturally occurring RNA. Site-specific cleavage (initial step) as well as in vitro recombination of fragments (last step) is performed by one of the methods outlined above ( Fig. 2 , 3 , and 5 ). Terminal nucleotide Ζ of the 5′-half RNA fragment can be quantitatively removed after periodate oxidation of the 2'-3′-glycol end followed by a β-elimination of the nucleoside induced by either aniline or lysine. The resulting 3 '-phosphate of the RNA fragment, now lacking nucleotide ZMP, is removed by phosphatase. The subsequent ligation with a large excess of nucleotide Y (in the form of 3′-5′-diphosphate) is catalyzed by T4 RNA ligase. The 5′-P of the newly incorporated donor (Y) dinucleotide phosphate can be radiolabeled with 32P (symbolized by an asterisk). Phosphatase treatment of this newly synthesized RNA molecule allows a new cycle of ligation reaction with an appropriate donor RNA molecule. The global yield of this stepwise synthesis of chimeric RNA is rather low (at best a few percent of the starting material). Alternative methods for site-specific introduction of modified nucleotides in RNA are detailed in Chapter 4 by Zimmermann et al. (see also Moore and Query, 1997).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 7

Schematic of a procedure for direct RNA sequencing of an RNA fragment containing modified nucleotides. The RNA is first cleaved randomly (single cut per molecule) under mild conditions. Each fragment is then radiolabeled with P at the 5′ end. The resulting 5′-radiolabeled fragments are separated by electrophoresis on a polyacrylamide gel. Hydrolysis of each fragment into mononucleotides is performed in situ with RNase T2. Contact transfer of the resulting nucleotides is performed on a PEI-cellulose plate. After the chromatography, the plate is autoradiographed. Identification of each radiolabeled terminal 3′,5′-dinucleotide phosphate on the plate is obtained from its position as compared with those of published reference maps (method of Gupta and Randerath, 1979). The presence of 2'-O-methylated nucleosides in RNA is signaled by “gaps” in the ladder on the gel. Alternatively, the RNA fragment from each band of the gel can be eluted and completely digested by nuclease P1, and the resulting 5′-nucleotide monophosphate can be analyzed by 2D thin-layer chromatography. Identification of each radiolabeled spot on the plate is performed by comparison with those of published reference maps ( ). The example illustrates the sequencing of the tRNA region corresponding to the ΤΨ-loop. This method could also be used to detect base-conversion-type RNA editing processes.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 8

Experimental protocol for the determination of the base composition of an RNA fragment, including modified nucleotides (designated X in the example). The series of steps ends up in 2D thin-layer chromatographic analysis of the 5′-P-nucleotide monophosphate (qualitative test). Identification of each radiolabeled spot on the plate is performed by comparison with those in published reference maps ( ). An alternative approach is based on quantitative analysis of the nucleoside composition by high-pressure liquid chromatography (HPLC) coupled to a diode array spectrophotometer (spectral analysis) and/or mass spectrometry (for details see Chapter 3 by Crain).

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 9

Nearest-neighbor analysis of modified nucleotides in RNA. The RNA fragment has to be internally labeled with P at one of the four nucleotides (in this example, 5′-P of CMP). The 2D thin-layer analysis of the nuclease P1 hydrolysate (that retains the 5′-phosphate) allows the identification of the modified nucleotides corresponding to the parent radiolabeled nucleotide (in the example of the figure, a 5′-CMP derivative posttranscriptionally modified on the base or sugar represented by C*). The chromatographic analysis of the nucleotides obtained after complete hydrolysis of the same radiolabeled RNA but with RNase T2 (leaving the 3′-phosphate) allows all of the modified nucleotides that are 3′-adjacent to a CMP in the original RNA chain (nearest neighbor) to be revealed.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 10

Identification of a C-to-U editing site by the primer extension assay. Here a 5′-P-labeled DNA primer (thick black line) is prepared so that it hybridizes close to the editing site, leaving no C residue between the 3′ end of the primer and the first putative edited C. Reverse transcription is performed in the presence of dATP, dTTP, dCTP, and ddGTP. A “strong stop” will appear on the sequencing gel at unedited C, except if an edited U is present ( ). The same analysis can be performed for testing α-to-I editing or modification sites in RNA. Indeed, inosine will be read as G during transcription. From such analysis, the ratio of edited to nonedited molecules at a given position in the RNA population can be evaluated.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 11

Detection of modified nucleotides in RNA by combined chemical and reverse transcription techniques. The cartoon illustrates different potential situations. In one case (left part) RNA contains a naturally occurring bulky residues. It will cause a strong stop upon reverse transcription that is easily detected on the sequencing gel after primer extension. In vitro chemical alteration of RNA [for example, with l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT)] can produce bulky residues (middle part of the figure) that will also generate strong stops on the sequencing gel. However, if the in vitro chemical conditions are optimized for producing random (partial) chemical “hypermodification,” several positions of the same RNA molecule will give the strong stop phenomenon. The right part of the figure illustrates the case where naturally occurring nucleotide modifications (like 2'-O-methylation of the ribose moiety) or an in vitro random chemical alteration (as with glyoxal on G) can render RNA resistant to enzymatic or hydrolytic cleavage at specific sites. These sites will be revealed on the sequencing gel by “gaps” in the radiolabeled RNA “ladder” after primer extension. This type of analysis gives only qualitative information, and cannot be used to quantify the relative amount of a modified nucleotide at a given position of an RNA population. The main advantages are that the RNA does not have to be pure, and that a long RNA can be explored with different primers complementary to different regions of the RNA molecule.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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Figure 12

Alternative protocols for the identification of enzyme activities corresponding to selected RNA modification or editing phenomena. For certain types of analysis, the RNA used as substrates has to be radiolabeled with P. This can be done either during in vitro or in vivo transcription of synthetic or natural genes, or during the recombination of RNA fragments. Details of the preparation of different substrates and RNA modification/editing analysis are described in the previous figures. HPLC, high-performance liquid chromatography; MS, mass spectrometry.

Citation: Grosjean H, Motorin Y, Morin A. 1998. RNA-Modifying and RNA-Editing Enzymes: Methods for Their Identification, p 21-46. In Grosjean H, Benne R (ed), Modification and Editing of RNA. ASM Press, Washington, DC. doi: 10.1128/9781555818296.ch2
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