Atlas of mRNA translation and decay for bacteria.

Regulation of messenger RNA stability is pivotal for programmed gene expression in bacteria and is achieved by a myriad of molecular mechanisms. By bulk sequencing of 5' monophosphorylated mRNA decay intermediates (5'P), we show that cotranslational mRNA degradation is conserved among both Gram-positive and -negative bacteria. We demonstrate that, in species with 5'-3' exonucleases, the exoribonuclease RNase J tracks the trailing ribosome to produce an in vivo single-nucleotide toeprint of the 5' position of the ribosome. In other species lacking 5'-3' exonucleases, ribosome positioning alters endonucleolytic cleavage sites. Using our metadegradome (5'P degradome) sequencing approach, we characterize 5'P mRNA decay intermediates in 96 species including Bacillus subtilis, Escherichia coli, Synechocystis spp. and Prevotella copri and identify codon- and gene-level ribosome stalling responses to stress and drug treatment. We also apply 5'P sequencing to complex clinical and environmental microbiomes and demonstrate that metadegradome sequencing provides fast, species-specific posttranscriptional characterization of responses to drug or environmental perturbations. Finally we produce a degradome atlas for 96 species to enable analysis of mechanisms of RNA degradation in bacteria. Our work paves the way for the application of metadegradome sequencing to investigation of posttranscriptional regulation in unculturable species and complex microbial communities.

Improved computational analysis of ribosome dynamics from 5'P degradome data using fivepseq.

In eukaryotes, 5'-3' co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus, 5' monophosphorylated (5'P) degradome sequencing, in addition to informing about RNA decay, also provides information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome; however, computational tools for their reproducible analysis are lacking. Here, we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5'P degradome data. This tool performs both metagene- and gene-specific analysis, and enables easy investigation of codon-specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene-specific ribosome pauses in Saccharomyces cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific degradation frameshifts. To show its wide applicability, we investigate 5'P degradome from Arabidopsis thaliana and discover both motif-specific ribosome protection associated with particular developmental stages and generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5'P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis.