Detection of Inosine on Transfer RNAs without a Reverse

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Detection of Inosine on Transfer RNAs without a reverse transcription reaction Adrian Gabriel Torres, Thomas Fabian Wulff, Marta RodríguezEscribà, Noelia Camacho, and Lluis Ribas de Pouplana Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00718 • Publication Date (Web): 10 Sep 2018 Downloaded from http://pubs.acs.org on September 11, 2018

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Biochemistry

Detection of Inosine on Transfer RNAs without a reverse transcription reaction Adrian G. Torres1,*, Thomas F. Wulff1†, Marta Rodríguez-Escribà1, Noelia Camacho1 and Lluís Ribas de Pouplana1,2,*. 1 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Parc Científic de Barcelona, C/ Baldiri Reixac 10, 08028 Barcelona, Catalonia, Spain. 2 Catalan Institution for Research and Advanced Studies (ICREA), P/ Lluis Companys 23, 08010 Barcelona, Catalonia, Spain. * Corresponding authors.

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ABSTRACT

Inosine at the ‘wobble’ position (I34) is one of the few essential posttranscriptional modifications in transfer RNAs (tRNAs). It results from the deamination of adenosine and occurs in Bacteria on tRNAArgACG and in Eukarya on 6 to 7 additional tRNA substrates. Because inosine is structurally a guanosine analogue reverse transcriptases recognizes it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (Splinted Ligation-based Inosine Detection), a reverse-transcription-free method to detect inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and PAGE analysis. We show that the method can detect I34 on different tRNA substrates, and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.


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INTRODUCTION Posttranscriptional modification of adenosine to inosine at position 34 (I34) of transfer RNAs (tRNAs) is the result of a deamination reaction catalyzed by adenosine deaminases. I34 is present in nearly all natural tRNAs genetically encoded with adenosine at this position (A34); i.e. tRNAArgACG in Bacteria (catalyzed by TadA) and tRNAArgACG, tRNAAlaAGC, tRNAProAGG, tRNAThrAGU, tRNAValAAC, tRNASerAGA, tRNALeuAAG, and tRNAIleAAU in Eukarya (catalyzed by hetADAT); while it is absent in Archea

1, 2

. Because inosine can base pair with adenosine,

cytidine and thymidine, I34 expands the number of codons A34-tRNAs can recognize and is therefore critical for gene translation. Consequently, knockouts of the enzymes responsible for I34 synthesis lead to lethality in all organisms studied thus far

3-8

, making I34 one of the few

essential posttranscriptional modifications on tRNAs described to date. Several methods to detect I34 exist. tRNAs can be radiolabeled and digested with RNases to single nucleotides which are then resolved by thin-layer chromatography

3, 4, 6

. Alternatively,

tRNAs can be first treated with glyoxal/borate to protect guanosine residues and then be digested with RNase T1, an enzyme that cleaves guanosine and inosine residues. Upon glyoxal protection only I34-containing tRNAs will be cleaved, and can be visualized by denaturing polyacrylamide gel electrophoresis (PAGE) 9. These approaches can be used to study A34-to-I34 deamination in vitro but cannot be easily applied to detection of I34 from in vivo derived samples. In order to detect I34 on tRNAs derived from cells, liquid chromatography coupled to mass spectrometry can be used

10

. However, this method is laborious, low-throughput, requires large amounts of

purified tRNAs and specialized equipment.


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Currently, strategies based on reverse transcription (RT) of tRNAs are the methods of choice for I34 detection on in vitro- and in vivo-derived samples. Reverse transcriptases recognize I34 as a guanosine (G34); hence, upon polymerase chain reaction (PCR) amplification of reverse transcribed products (RT-PCR), amplicons can be sequenced and the ratio of A34-to-G34 conversion can be calculated

5, 9

. Because sequencing makes this approach expensive and low-

throughput, restriction fragment length polymorphism (RFLP) analyses can be performed as an alternative, taking advantage of the A-to-G conversion on PCR amplicons that either create or abolish sites for cleavage by specific restriction enzymes

11

. While these RT-based methods for

I34 detection have proven useful in the past, a major limitation they all present is that other posttranscriptional modifications on tRNAs can bias or even block the RT reaction 12, 13, and thus potentially abolish detection of I34 on certain tRNA species. In this sense, there is still a need for an RT-free strategy for I34 detection that could be applied in a straight forward and costeffective manner to small amounts of in vitro- and in vivo-derived samples as starting material, and that could be performed at a medium- to high-throughput scale.

DEVELOPMENT OF THE METHOD In recent years conflicting evidence has emerged regarding the I34 modification status of tRNAArgACG in higher Eukaryotes

14

. While I34 could be readily detected by sequencing of

tRNAArgACG in Escherichia coli, Mycoplasma capricolum, and Saccharomyces cerevisiae; it could not be detected in nematodes, mosses, higher plants, insects, and mammals 14-18. However, the presence of I34 on human tRNAArgACG has been documented by other means

19

; suggesting

that at least in some of these cases RT-based methods may be leading to artifactual results.


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Biochemistry

To test this hypothesis, we attempted to detect I34 on tRNAArgACG using two RT-based methods: standard Sanger sequencing and RFLP. Figure 1 shows that while I34 can be efficiently detected in in vitro transcribed human tRNAArgACG upon in vitro deamination with human hetADAT (Figure 1A-B), it cannot be detected in the in vivo-derived endogenous tRNAArgACG from HeLa cells (Figure 1C-D). Thus, we set out to develop an RT-free method for I34 detection.

Figure 1. RT-dependent methods cannot detect I34 on in vivo-derived human tRNAArgACG. (A) Sanger sequencing profiles of RT-PCR amplicons derived from in vitro transcribed tRNAArgACG before (upper panel) and after (lower panel) treatment with purified human hetADAT. The sequence corresponding to the tRNA anticodon is underlined and the ‘wobble’ residue (position 34 of the tRNA) is indicated with an asterisk. Unmodified A34 is detected as an “A”, while I34 is detected as a “G” at this position. (B) Detection of I34 from the same amplicons as in (A)


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using RFLP. Schematic representation of the recognition site for AciI (upper panel), which will cleave amplicons derived from I34-containing tRNAArgACG. A representative RFLP agarose gel is shown (lower panel). Sanger sequencing spectrum (C) and RFLP (D) of RT-PCR amplicons derived from endogenous human tRNAArgICG from HeLa cells. I34 is not detected.

In general, splinted-ligations are used to ligate a DNA oligonucleotide

32

P-radiolabeled “ligation”

(3’-Ligation DNA) to the 3’-end of a small RNA of interest (5’-RNA)

containing a free 3’-hydroxyl (3’-OH) group; resulting in an RNA:DNA chimeric species

20-22

.

First a DNA “bridge” oligonucleotide is designed so that its 3’-half is perfectly complementary to the 5’-RNA, and its 5’-half is perfectly complementary to the 3’-Ligation DNA. The three species are then hybridized together so as to create a double stranded structure with a nick on one strand (leaving the 3’-OH of the 5’-RNA in near proximity to the 5’-phosphate of the 3’-Ligation DNA) (“Capture Reaction”). The 5’-RNA and the 3’-Ligation DNA are then joined by incubation with T4 DNA ligase. This ligation product (RNA:DNA chimera) can be visualized by PAGE and autoradiography. Endonuclease V is a repair enzyme with wide substrate specificity on DNA. It recognizes xanthine, hypoxanthine, uracil, base mismatches, apurinic/apyrimidinic sites, unpaired loops, hairpins, 5’-flaps, and pseudo-Y structures

23, 24

. Endonuclease V can also specifically cleave

single stranded inosine-containing RNAs, generating a 5’ RNA fragment with a free 3’-OH group

19, 25

. We took advantage of this activity to develop SL-ID (Splinted Ligation-based

Inosine Detection) (Figure 2). In this method, total RNA is treated with endonuclease V (Step 1, Figure 2), and the resulting I34-containing-5’-arm of the tRNA is then captured by a specific


6

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Biochemistry

DNA bridge oligonucleotide that also shares complementarity with a

32

P-ligation DNA

oligonucleotide (Step 2, Figure 2). The tRNA half and the ligation oligonucleotide are then ligated by the T4 DNA ligase (Step 3, Figure 2). Importantly, in the absence of I34, the tRNA is not cleaved by endonuclease V and no productive ligation is possible. After the ligation reaction, samples are subjected to phosphatase treatment to remove the radioactive mark on the remaining 3’-Ligation DNA, preserving the internally-labelled

32

P

ligation product (Step 4, Figure 2). Finally, samples are resolved by denaturing PAGE where the expected RNA:DNA chimera resulting from productive ligation is resolved from remaining traces of the 32P-labelled 3’-Ligation DNA (Step 5, Figure 2). All other non-radioactive species resulting from these treatments (full length tRNA, 3’-half of cleaved tRNA, bridge oligonucleotide and 3’-Ligation DNA) are not detected. Because the length of the 5’-half of most tRNAs is similar in size (~35 nt-long), the expected ligation product regardless of the tRNA species or the organism where it derives from, will usually be ~49 nt-long (35 nt of 5’-tRNA half plus 14 nt of 3’-Ligation DNA). Note however, that the ligation product obtained by SL-ID migrate at a slightly higher molecular weight (~ 55 nt) probably due to its RNA:DNA chimeric nature (see below).


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Figure 2. Schematic representation of SL-ID. 1) Total RNA is treated with endonuclease V leaving a free 3’-hydroxyl group (-OH) on the 5’-I34-containing half of the tRNA. 2) A DNA bridge oligonucleotide (oligo) captures the 5’-arm derived from the I34-tRNA after endonuclease


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Biochemistry

V cleavage and the

32

P-ligation DNA oligo. 3) The cleaved I34-tRNA half is ligated to the

ligation DNA oligo (green arrow). 4) A phosphatase removes the radioactive label (red circle) of unligated ligation DNA oligo (some free

32

P-ligation DNA oligo remains). 5) Samples are

resolved by denaturing polyacrylamide gels (PAGE) where only the I34-tRNA-half:32P-ligation DNA oligo chimera and the free

32

P-ligation DNA oligo are detected. When the tRNA is

unmodified (right panels) no productive ligation is obtained and only free

32

P-ligation DNA

oligo is observed by PAGE. RNA species are depicted in black and DNA species are depicted in blue.

VALIDATION OF THE METHOD Detection of endogenous I34 on human tRNAs by SL-ID. We have previously shown that among all human substrates of hetADAT, tRNAValAAC is the one where I34 is most easily detected by sequencing 5. We therefore used this substrate to initially verify the performance of SL-ID. Figure 3A shows the expected I34-containing RNA:DNA chimera for the in vitro transcribed tRNAValAAC treated with hetADAT, but not for its unmodified A34-containing counterpart (Figure 3A). This indicates that the presence of I34 is required to obtain a productive ligation by SL-ID. Moreover, SL-ID could also detect I34 in endogenous human tRNAValAAC derived from two different cell lines: HEK293T (Figure 3A) and HeLa (Figure 3B) cells. Given the difficulty in detecting I34 on human tRNAArgACG by RT-based methods (Figure 1) 14

5,

, we next applied SL-ID to verify the presence of I34 on this tRNA in HEK293T cells. We

found that A34 is modified to I34 on this tRNA, and that the modified base is efficiently and specifically identified by SL-ID (Figure 3C). Similar results were obtained using total RNA


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from HeLa cells, where we applied SL-ID to detect I34 on tRNAArgACG, tRNAValIAC, tRNAThrIGU and tRNAAlaIGC (Figure 3D). We also verified the specificity of the DNA bridge oligonucleotides for their corresponding tRNAs. I34 could be detected on in vitro transcribed tRNAArgACG treated with hetADAT using BridgeArgICG, but not with BridgeValIAC (Figure 3E). Likewise, I34 could be detected on in vitro transcribed tRNAValAAC treated with hetADAT using BridgeValIAC, but not with BridgeArgICG (Figure 3F). Additionally, these experiments show that the ligation products obtained with pure transcripts of tRNAArgICG and tRNAValIAC are equivalent to those obtained with RNA purified from cell lines (Figure 3A-D). Altogether, these results show that SL-ID overcomes the limitations of RT-based methodologies for I34 detection, and can be applied to different I34-containing tRNA substrates, in different cell types or in in vitro-derived samples.


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Biochemistry

Figure 3. Detection of I34 by SL-ID on human in vitro and in vivo-derived tRNAs. (A) SL-ID using a bridge oligonucleotide to capture human tRNAValIAC (Bridge

Val

IAC).

Productive ligation

is obtained for in vitro transcribed tRNAValAAC only when it is modified by the human heterodimeric adenosine deaminase acting on tRNA (hetADAT), and is treated with endonuclease V (Endo V). I34 is also detected on endogenous tRNAValIAC from HEK293T cells


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in an Endo V-dependent manner. (B) Experiment as in (A) against endogenous tRNAValIAC from HeLa cells. (C) Detection of I34 on endogenous tRNAArgACG from HEK293T cells. Productive ligation is not obtained in the absence of bridge oligo (Bridge

Arg

ACG)

or in the absence of total

RNA. (D) Detection of I34 on endogenous tRNAValIAC, tRNAArgICG, tRNAThrIGU, and tRNAAlaIGC from HeLa cells. (E) Detection of I34 on in vitro transcribed tRNAArgACG upon treatment with hetADAT. Productive ligation is not observed when using a BridgeValIAC oligonucleotide. (F) Detection of I34 on in vitro transcribed tRNAValAAC upon treatment with hetADAT. Productive ligation is not observed when using a BridgeArgICG oligonucleotide. Note that partial tRNA degradation can sometimes occur during the processing of the samples, and may result in the formation of ligation products of a smaller size that migrate at molecular weights below the expected ~ 55 nt (e.g. panels C-D: smear on samples derived from total RNA; panels E-F: discrete cleavage sites for in vitro transcribed tRNA samples).

Detection of endogenous I34 on tRNAArgACG in murine tissues by SL-ID. We next wanted to verify that SL-ID can also be applied to RNA extracted from different tissues and in a different organism. Thus, we evaluated detection of I34 on murine tRNAArgACG in 4 different tissues. Productive ligation by SL-ID was observed in all analyzed samples (Figure 4) indicating that I34 is present on this tRNA in mice.


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Biochemistry

Figure 4. Detection of I34 on tRNAArgACG in murine tissues by SL-ID. Mm: Mus musculus.

DISCUSSION I34 is an essential posttranscriptional tRNA modification of biomedical importance that played an important role in the evolution of eukaryotic genomes and proteomes2

26, 27

. Currently

available methods to detect I34, such as RT-based sequencing strategies, can be limited by technical artifacts that compromise their interpretation 14 (Figure 1). We have previously shown that deep sequencing of tRNAs can be used to detect I34 on precursor tRNAs in vivo, but the efficiency of I34 detection varied considerably among different I34-tRNA species. For example, while ~ 90 % of sequencing reads mapping to tRNAValAAC reflected the presence of I34, less than 2 % of reads mapping to tRNAArgACG evidenced I34 modification on this tRNA 5. This is consistent with other reports that describe the difficulty of detecting I34 on tRNAArgACG in higher eukaryotes by RT-based methods 14. The exact mechanisms that cause these RT-based artifacts are unknown. We speculate that other tRNA modifications may block the reverse transcription of certain tRNAs. For example, position


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37 is modified to 1-methylguanosine in murine tRNAArgACG, where I34 cannot be easily detected by RT-based methods 14. In contrast, this residue is unmodified in yeast tRNAArgACG, and I34 is readily detected in this tRNA

14

(Supporting Information Figure S1)

28

. Similarly, I34 is

detected by RT-based methods in tRNAValAAC 5, the only eukaryotic I34-containing tRNA consistently lacking modifications at position 37 (Supporting Information Figure S1). Other modifications at different positions may also generate RT-artifacts to different extents. SL-ID (Figure 2) is a method that can verify the presence of I34 when RT-based methods are unable to (Figure 3). In addition, while RT-based techniques detect I34 mainly on precursor tRNAs 5, 11, SL-ID detects I34 on mature tRNAs (the observed ligation product is consistent with 5’-tRNA halves after precursor tRNA processing) (Figure 3 and Figure 4). We have applied this approach to both in vitro- and in vivo-derived I34-tRNAs, including those derived from different human cell lines or murine tissues (Figure 3 and Figure 4). We show here that i) I34 is necessary and sufficient for detection by SL-ID (Figure 3A); ii) detection is strictly dependent on cleavage by endonuclease V (Figure 3 and Figure 4); and iii) productive ligation does not occur in the absence of RNA or bridge oligonucleotide (Figure 3B). Highly specific oligonucleotide probes are an essential condition for all hybridization-based techniques, including RT-based methods for which specific primers are needed. Thus, a key step to successfully use SL-ID is the design of a bridge oligonucleotide highly specific for the tRNA sequence of interest (Figure 3E and Figure 3F). I34 can also be detected by northern blotting after endonuclease V treatment

19

. However,

comparative analyses revealed that splinted ligation on small RNAs can be up to 50 fold more sensitive than northern blotting using DNA probes 21. In addition, splinted ligation protocols do


14

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Biochemistry

not require transfer of the RNA from gels to membranes, additional hybridization of radioactive probes, or washing steps. Finally, probes used for northern blotting can bind equally well to precursor-tRNAs, mature tRNAs and tRNA-derived fragments because they share the same nucleotide sequence, independently of the presence of I34. By contrast, SL-ID only detects I34containing tRNAs possessing a free 3’-OH group after endonuclease V cleavage, thus avoiding detection of other non-I34-containing tRNA and tRNA-derived species. While splinted ligation can be quantitative

21

, we would like to emphasize that SL-ID as

presented here is not a quantitative assay. This is mainly due to the relatively low efficiency of I34 cleavage by bacterial endonuclease V, which was shown to be less efficient at cleaving inosine-containing single stranded RNA than eukaryotic endonuclease V

19, 25

. For practical

reasons, we have developed SL-ID using a commercially available Thermotoga maritima endonuclease V, however a high-efficiency enzyme may allow a quantitative use of the method. There are several instances where non-quantitative detection of I34 by SL-ID can be beneficial. We have applied the method to shed light on an ongoing controversy regarding the modification status of mammalian tRNAArgACG 14; and have shown that in both humans and mice, this tRNA is modified to I34 (Figure 3 and Figure 4). Likewise, SL-ID can provide a direct approach to address the modification status of tRNAs from less studied species across all domains of life (Rafels-Ybern et al manuscript in preparation).

STEP BY STEP DESCRIPTION OF SL-ID 200 ng of in vitro transcribed tRNA or 2 µg of total RNA were digested with endonuclease V (Thermo Scientific) overnight at 37 ºC following the manufacturer’s protocol in a reaction


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volume of 25 µL. Endonuclease V reactions were then purified using the MiTotal RNA Mini extraction kit (Viogene; Catalog Nº VTR1001). RNA was eluted from the purification column with 50 µL H2O and was precipitated with ethanol. The obtained RNA pellet (often invisible) was washed once with 1 mL of ethanol 75 % and resuspended in 10 µL H2O. The expected ~ 20 ng/µL RNA concentration for in vitro transcribed RNA or ~ 200 ng/µL RNA concentration for total RNA was confirmed using a Nanodrop ND-1000. tRNA-specific DNA bridge oligonucleotides were used to capture tRNAValIAC [5’GAATGTCATAAGCGTCAGGCGAACGTGATAACCACTACAC-3’],

tRNAAlaIGC

GAATGTCATAAGCGGCAGTCAGACGCGTTATCCATTG-3’], GAATGTCATAAGCGCCAAGCGAGCGCTCTACCATTTG-3’]

tRNAArgICG

and

[5’[5’-

tRNAThrIGU

[5’-

GAATGTCATAAGCGCCAGACAGGCGCTTTAACCAACTAAG-3’]. A bridge oligonucleotide working solution was prepared in 10X Capture Buffer (100 mM TrisHCl pH 7.5; 750 mM KCl) to a final concentration of 100 nM. 1.5 µL of 100 µM Ligation DNA oligonucleotide

[5’-CGCTTATGACATTC-3’]

22

was

5’-end

radiolabeled

with

T4

Polynucleotide Kinase (Takara; Code Nº 2021A) following the manufacturer’s protocol. 9 µL of endonuclease V-digested RNA (~ 180 ng or 1.8 µg for in vitro transcribed or total RNA, respectively) were hybridized with 1.1 µL tRNA-specific bridge oligonucleotide [100 nM] and 1 µL

32

P-labeled ligation oligonucleotide. Samples were heated at 95 ºC for 5 minutes and were

left at room temperature overnight to slowly cool down for efficient hybridization. Note: Efficient hybridization can also be obtained by leaving the samples at room temperature for 2 hours.


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1 µL T4 DNA Ligase (Fermentas; Catalogue Nº EL0335), 1.5 µL T4 DNA Ligase Buffer 10X (Fermentas) and 2.5 µL H2O was added to each reaction (final volume 16 µL) and incubated for 1 hour at 37 ºC. After incubation, 1 µL Calf Intestine Phosphatase (New England Biolabs; Catalogue Nº M0290S) was added to each reaction to remove the 5’-end

32

P from free ligation

oligonucleotide and samples were incubated at 37 ºC for 1 hour. Enzymes were heat inactivated at 95 ºC for 5 minutes and samples were stored at -20 ºC until ready to use. 4 µL of tRNA loading buffer (8M urea, 30 % glycerol; 20 % formamide; bromophenol blue/xylene cyanol) were added to each reaction, and 20 µL of sample loaded into a 12 % polyacrylamide gel containing 8 M urea. Samples were run for 1.5 hours at 120V; and the gels were directly exposed to a Typhoon Screen for 30 minutes. Radioactive signals were detected with a Typhoon Scanner. Optional step: If the radioactive signal is not strong enough to be detected after 30 minutes of gel exposure, we recommend drying the gel for 1 hour at 80 ºC using a Slab Gel Dryer GD2000 before exposing it to a Typhoon Screen, to prevent diffusion of the radioactive signal. In our hands the full SL-ID protocol can be performed in less than two days.

ADDITIONAL EXPERIMENTAL PROCEDURES In vitro transcribed tRNAs (Supporting Information Table S1) were obtained and in vitro deaminated with purified human hetADAT as previously described HEK293T cells was purified as previously described

5, 11

11

. RNA from HeLa and

. RNA from murine tissues was a kind

gift from Dr Joan Ginovart’s lab (IRB Barcelona) 29. Sanger sequencing and RFLP experiments were performed as previously described 11. PCR amplification of in vitro transcribed tRNAArgACG


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was performed with primers “Hs tRNA Arg ACG, oligo 1” and “Hs tRNA Arg ACG, oligo 6” (Supporting Information Table S1) using an annealing temperature of 60 ºC. PCR amplification of endogenous tRNAArgICG from HeLa cells was performed with primers “FWD tRNA Arg ACG” and “RVR tRNA Arg ACG” (Supporting Information Table S1) using an annealing temperature of 63 ºC. Treatments with AciI (New England Biolabs; Catalog Nº R0551S) were performed for 4 hours at 37 ºC.

ASSOCIATED CONTENT Supporting Information Available. Pattern of modified residues on I34-containing tRNAs based on the tRNAmodViz database. Oligonucleotides used for in vitro transcription of tRNAs, Sanger sequencing, and RFLP experiments. This material is available free of charge (file type, PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (AGT). Tel: +34934034867. Fax: +34934034870 *Email: [email protected] (LRdP). Tel: +34934034868. Fax: +34934034870 Present Addresses †T.F.W: Max-Planck-Institut fur Infektionsbiologie. Charitéplatz 1. Campus Charité Mitte Berlin, DE 10117. Author Contributions


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Biochemistry

A.G.T. and T.F.W. performed experiments. A.G.T., T.F.W. and L.R.dP. conceived the project. M. R.-E. and N.C. performed purification of hetADAT and in vitro transcription of tRNAs. A.G.T. and L.R.dP. wrote the manuscript with contributions from all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO201564572-R and BIO2014-61411-EXP to L.R.dP). Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We would like to thank the Ribas de Pouplana lab for helpful discussions and Dr Joan Ginovart’s lab (IRB Barcelona) for providing total RNA from mouse tissues.

ABBREVIATIONS tRNA, transfer RNA; I34, inosine at position 34 of the tRNA; A34, adenosine at position 34 of the tRNA; TadA, tRNA adenosine deaminase A; ADAT, adenosine deaminase acting on tRNA; hetADAT, heterodimeric ADAT; RFLP, restriction fragment length polymorphism; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; PCR, polymerase chain reaction; 3’-OH, 3’-hydroxyl.


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Detection of Inosine on Transfer RNAs without a reverse transcription reaction Adrian G. Torres1,*, Thomas F. Wulff1†, Marta Rodríguez-Escribà1, Noelia Camacho1 and Lluís Ribas de Pouplana1,2,*.


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Detection of Inosine on Transfer RNAs without a Reverse

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