Abstract
2-Methylthio-N6-isopentenyladenosine (ms2i6A) is an evolutionally conserved posttranscriptional modification found at position 37 of four mammalian mitochondrial tRNAs, mt-tRNASer(UCN), mt-tRNATrp, mt-tRNAPhe and mt-tRNATyr. The ms2 modification in ms2i6A strengthens codon-anticodon interaction and contributes to accurate and efficient decoding. Deficiency of ms2 modifications impairs mitochondrial protein synthesis, which ultimately leads to the development of myopathy in mice and patients having mitochondrial diseases. Therefore, the level of ms2 could be utilized as an indicator that reflects the status of mitochondrial protein synthesis. Here, we describe a simple and fast quantitative PCR-based method to measure the ms2 level in total RNA sample.
Keywords: TRNA, Modification, Mitochondria
Materials and Reagents
Equipment
Procedure
Part I. DNase treatment
Part II. Reverse transcription Figure 1. Workflow of the method to detect ms2 modification. The mitochondrial tRNA is reversely transcribed by r1 primer and r2 primer, respectively. Because of the inhibitory effect of ms2-modification to the reverse transcription, the amount of cDNA generated by r2 primer (Green lines) highly depends on the ms2 levels in a given RNA sample. On the other hand, the cDNA generated by r1 primer (Blue lines) is independent of ms2 level, and could be utilized as an internal control. The amount of each cDNA is quantified by a subsequent quantitative PCR (qPCR) using f1 and r1 primers.
Part III. Quantitative PCR
Part IV. Data analysis Analyze data from Samples (r1) and (r2) to obtain Ct values. The results obtained Samples (r1) and (r2) represents the total tRNA level and ms2-modification level in individual tRNA, respectively. The normalized modification level in any given RNA sample is calculated as dCt = Ct (r2) – Ct (r1). Because the dCt value precisely reflects the modification level (Xie et al., 2013), the dCt value could be directly used for comparison of modification levels between multiple samples (see below for the representative data).
Representative data
Figure 2. Measurement of ms2 level of mt-tRNATyr in wild-type mouse heart tissue. Total RNA was purified from mouse heart tissue using TRIzol followed by 2-propanol precipitation. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (green) and r2 primer (blue) are shown. Ct (r2) = 14.8, Ct (r1) = 10.7. Modification level (dCt) in wild-type mouse heart = 14.8-10.7 = 4.1. Figure 3. Measurement of ms2 level of mt-tRNATyr in heart tissue of Cdk5rap1 KO mouse. Total RNA was purified from heart tissue of Cdk5rap1 KO mouse that does not contain ms2 modification. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (red) and r2 primer (blue) are shown. Ct (r2) = 10.4, Ct (r1) = 10.4. Modification level (dCt) in Cdk5rap1 KO mouse heart = 10.4-10.4 = 0. Figure 4. Comparison of the modification levels of mt-tRNATyr in heart tissues of wild-type (WT) and Cdk5rap1 KO mouse
Acknowledgments
The concept of this protocol was adapted from our previous study, in which the quantitative PCR was used to measure ms2 modification in cytosolic tRNALys(UUU) (Xie et al., 2013). We have successfully used this protocol to measure ms2 modification mitochondrial tRNA in our recent study (Wei et al., 2015). This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References
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