RNAseq of Bacillus subtilis with different tailing methods

Bacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashorttranscripts (up to 14 nt), termed product RNAs (pRNAs), that play a key role in reversingthe blockage of RNAP by 6S RNA. Here we resolved the pRNA length profile of 6S-1 RNAfrom Bacillus subtilis, a major model system for the study of 6S RNA biology, duringoutgrowth of cells from extended stationary phase. 9-mers were found to be a particularlyabundant pRNA length species, followed by 8/10/11-mers and 13/14-mers. Consistent with invitro data from the Escherichia coli system, these findings support the mechanistic modelaccording to which the housekeeping sigma factor (sig70 or sigA) dissociates from 6SRNA:RNAP complexes upon synthesis of pRNA 9-mers, followed by final dissociation of 6SRNA and RNAP upon synthesis of longer pRNAs (13/14-mers). Methodologically, theidentification of such ultrashort RNAs in total cellular extracts for RNA-Seq is inefficient withstandard protocols using adapter ligation to RNA 3'-ends for reverse transcription and PCR-based cDNA sequencing. Here, we demonstrate that ultrashort RNAs can be much moreefficiently incorporated into RNA-seq libraries by poly(A), poly(C) and potentially also poly(U)tailing of their 3'-ends. A straightforward approach that integrates RNA-seq results obtainedfrom different 3'-end tailings [poly(A,C,U)] in regions of homodimeric or homooligomericnucleotide stretches allows one to approximate the fraction of read counts at positions thatremain unresolved in 3'-tailings with only type of nucleotide. Finally, the awareness ofmethodological biases and potential applications of our approach to other short RNAs arediscussed.