Abstract
Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.
Highlights
Studies about RNA modification dynamics in human RNAs are among the most controversially discussed
While tRNA modifications are accessible for potential RNA erasers, rRNA modifications are placed in the functional regions of the ribosome[25]
We have recently developed nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) in bacteria[29,30] and yeast[31], which reveals the dynamics of RNA modification processes
Summary
Studies about RNA modification dynamics in human RNAs are among the most controversially discussed. We present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture. Current studies of RNA modifications are limited to either mass spectrometric analysis[16] or sequencing[27,28] Both techniques provide information on the modification status at the time point of sample harvest and give no details on the mechanisms of RNA modification adaptation. To overcome this limitation, we have recently developed NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in bacteria[29,30] and yeast[31], which reveals the dynamics of RNA modification processes. By combining differentially labeled media in a pulse chase set-up, we recently succeeded to observe tRNA demethylation through
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