New insights into microbial gene expression

A significant advantage of nanopore technology is the facility to sequence entire RNA transcripts in single reads. Furthermore, in addition to offering both cDNA and amplification-free direct-cDNA sequencing approaches, nanopore technology also uniquely enables direct sequencing of native RNA, allowing characterisation of base modifications alongside nucleotide sequence.

Grünberger et al.1 demonstrated how full-length, direct nanopore RNA sequencing reads provided novel insights into the transcriptomes of three diverse prokaryotes, namely, the bacterium Escherichia coli and the archaea Haloferax volcanii and Pyrococcus furiosus.

As prokaryotic RNA molecules lack or exhibit much shortened poly(A) tails to those found in eukaryotes, the team enzymatically polyadenylated the extracted RNA to allow compatibility with the direct sequencing workflow. Subsequent sequencing on the MinION revealed that 3’ UTR length distributions, which ranged from 30–70 nucleotides, were comparable for all three microorganisms studied (Figure 1). Conversely, the 5’ UTRs were significantly shorter in the two archaeal species, which, according to the researchers, supports the idea that posttranscriptional regulation is mediated via the 3’ rather than 5’ UTR in archaea (Figure 1).

The archaeal species also showed overrepresentation of poly(U) stretches at transcription termination sequences (TTS). Interestingly, H. volcanii and P. furiosus differed both in the number of poly(U) stretches and the number of uridine bases in each stretch.

The long direct RNA reads also provided novel insights into the poorly understood archaeal ribosomal RNA (rRNA) maturation pathway, including the detection of putative novel rRNA processing intermediates.

‘…direct RNA sequencing can be a useful tool to approach intricated maturation pathway like rRNA maturation, and expand our understanding of RNA maturation in prokaryotes’1

Finally, the hyperthermophile P. furiosus was shown to exhibit the greatest amount of rRNA base modifications, followed by E. coli and then H. volcanii. These results are in agreement with previous studies, which have suggested that base modifications may stabilise the rRNA molecules, especially at high temperatures. As a result, the levels of such modifications may reflect the organisms’ natural environmental conditions. According to the researchers, ‘Due to the extraordinary read length and the sensitivity to base modifications, Oxford Nanopore-based native RNA-seq can provide valuable insights into (r)RNA processing, (r)RNA modifications patterns and the transcription of large operons’ 1.

Figure 1: Comprehensive characterisation of transcript boundaries. a) Comparison of 5’UTR lengths showed good correlation between both nanopore sequencing and traditional short-read sequencing technology for all organisms; however, nanopore sequencing does not require specialised sample preparation (i.e. terminator exonuclease (TEX) treatment). Notably, both archaeal organisms exhibited significantly shorter 5’UTRs. b) 3’UTR length was comparable between all organisms studied. The nanopore E. coli and H. Volcanii data set was consistent with published short-read data. Image courtesy of Felix Grünberger, University of Regensburg, Germany.

1. Grünberger, F. et al. Nanopore-based native RNA sequencing provides insights into prokaryotic transcription, operon structures, rRNA maturation and modif