6S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase

TL;DR

This study identifies 9-mers as a key RNA product during Bacillus subtilis recovery from stationary phase and introduces a new RNA-Seq method to detect ultrashort RNAs.

Contribution

A novel RNA-Seq method using poly-tailing to detect ultrashort RNAs and the discovery of 9-mers as a prominent pRNA species in B. subtilis.

Findings

9-mers are the most abundant pRNA length species during Bacillus subtilis outgrowth from stationary phase.

Standard RNA-Seq protocols are inefficient for detecting ultrashort RNAs, but poly-tailing improves detection.

An algorithm combining data from multiple tailing methods can approximate RNA read counts at ambiguous positions.

Abstract

Bacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashort transcripts (up to ~14 nt), termed product RNAs (pRNAs), that play a key role in reversing the blockage of RNAP by 6S RNA. Here, we resolved the pRNA length profile of 6S-1 RNA from B. subtilis, a major model system for the study of 6S RNA biology, during outgrowth of cells from extended stationary phase. 9-mers were found to be a particularly abundant pRNA length species, followed by 8-/10-/11-mers and 13-/14-mers. Consistent with in vitro data from the Escherichia coli system, these findings support the mechanistic model according to which the housekeeping sigma factor (σ70 or σA) dissociates from 6S RNA:RNAP complexes upon synthesis of pRNA 9-mers, followed by final dissociation of 6S RNA and RNAP upon synthesis of longer pRNAs (13-/14-mers). Methodologically, the identification of such ultrashort RNAs in total cellular extracts by RNA-Seq is inefficient with standard protocols using adapter ligation to RNA 3’-ends for reverse transcription and PCR-based cDNA sequencing. Here, we demonstrate that ultrashort RNAs can instead be incorporated into RNA-Seq libraries by polyA-, polyC- and potentially also polyU-tailing of their 3’-ends. At positions where a non-tailing nucleotide is followed by one or more tailing nucleotides, an algorithm that integrates RNA-Seq results from at least two different 3’-end tailings allows one to approximate the fraction of read counts at such ambiguous positions. Finally, methodological biases and potential applications of our approach to other short RNAs are discussed.

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