Abstract
Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.
Highlights
The genome of Escherichia coli supports the transcription of four different types of RNA: ribosomal RNA, transfer RNA, messenger RNA and small non-coding RNA. rRNA and tRNA represent by far the most abundant and resilient RNA species
Many nucleotide analogs commonly used to label nucleic acids in vivo are antimetabolites that are detected by click- or immuno-reactions
Because the detection threshold is adjusted by modulating array exposure times, SPOt is perfectly adapted to profile low abundant species such as tRNAs associated with polysomes
Summary
The genome of Escherichia coli supports the transcription of four different types of RNA: ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA and small non-coding RNA (sRNA). rRNA and tRNA represent by far the most abundant and resilient RNA species. The genome of Escherichia coli supports the transcription of four different types of RNA: ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA and small non-coding RNA (sRNA). RRNA and tRNA represent by far the most abundant and resilient RNA species. The 5S rRNA (120 nucleotides) and the 23S rRNA (2,904 nucleotides) are essential components of the large ribosomal subunit whereas the 16S rRNA (1,542 nucleotides) is part of the small subunit. Eighty-six intron-less tRNA genes are transcribed into 48 unique tRNA sequences organized in 40 different tRNA isoacceptor families (tRNAs with different anticodons) [1]. These tRNAs are transcribed from one to six gene copies all encoding the characteristic CCA 3’ -end.
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