# 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.

## Key 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.