Abstract
The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.
Highlights
IntroductionMore than 140 chemical RNA modifications are known[1]. Chemical RNA modification expand the repertoire of four natural nucleosides and influence RNA structure and function
To date, more than 140 chemical RNA modifications are known[1]
We found that the m1A is present solely in transfer RNA (tRNA) and that genomic HIV-1 RNA, neither 7SL RNA do not contain this modification
Summary
More than 140 chemical RNA modifications are known[1]. Chemical RNA modification expand the repertoire of four natural nucleosides and influence RNA structure and function. There is growing evidence that coding messenger RNA (mRNA) and regulatory RNA contain various chemical modifications[2]. Their existence in mRNA is complicated to be proved by common LC/MS technique but special and selective profiling or capturing techniques for various RNA modification have been developed lately. The NAD captureSeq.[7] allowed identification of RNAs with covalently attached Nicotinamide adenine dinucleotide (NAD) at 5′ end This method has enabled the detection of NAD in sRNA, and mRNA in E. coli[7] and in eukaryotes[8,9]. The problem of immunoprecipitation methods is that they do not allow for single base resolution of particular RNA modification. The m6A editing and the recruitment of YTHDF proteins were identified as the positive regulators of HIV-1 mRNA expression
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