Repurposing Ribo-Seq to provide insights into structured RNAs

Abstract

8Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. 9 However, this method can enrich RNAs that are not bound by ribosomes, but rather, are 10 protected from degradation in another way. For example, Escherichia coli Ribo-Seq libraries 11 also capture reads from most non-coding RNAs (ncRNAs). These fragments of ncRNAs pass all 12 size selection steps of the Ribo-Seq protocol and survive hours of MNase treatment, presumably 13 without protection from the ribosome or other macromolecules or proteins. Since bacterial 14 ribosome profiling does not directly isolate ribosomes, but instead uses broad size range cutoffs 15 to fractionate actively translated RNAs, it is understandable that some ncRNAs are retained after 16 size selection. However, how these 'contaminants' survive MNase treatment is unclear. Through 17 analyzing metaRibo-Seq reads across ssrS, a well established structured RNA in E. coli, and 18 structured direct repeats from Clustered Regularly Interspaced Short Palindromic Repeats 19 (CRISPR) arrays in Ruminococcus lactaris, we observed that these RNAs are protected from 20 MNase treatment by virtue of their secondary structure. Therefore, large volumes of data 21 previously discarded as contaminants in bacterial Ribo-Seq experiments can, in fact, be used to 22 gain information regarding the in vivo secondary structure of ncRNAs, providing unique insight 23 into their native functional structures. 25Importance 26 We observe that 'contaminant' signals in bacterial Ribo-Seq experiments that are often 27 disregarded and discarded, in fact, strongly overlap with structured regions of ncRNAs. 28 Structured ncRNAs are pivotal mediators of bioregulation in bacteria and their functions are 29 often reliant on their specific structures. We present an approach to access important RNA 30 structural information through merely repurposing 'contaminant' signals in bacterial Ribo-Seq 31 experiments. This powerful approach enables us to partially resolve RNA structures, identify 32 novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large-33 scale and in vivo. 34 35 Observation 36Ribosome profiling (Ribo-Seq) in bacteria is a method that enriches for ribosome-37 protected RNAs and therefore, enables the study of active translation events 1,2 . However, this 38 enrichment is not highly selective for ribosomes as relevant protocols do not specifically isolate 39 ribosomes, but rather select for them within an expected size range. Ribo-Seq is especially 40 challenging in bacteria because, unlike in yeast and eukaryotes, bacteria have a broad size 41 distribution of ribosome-protected footprints, ranging from 15-40 nucleotides 3 . The size range 42 that should be selected can vary across Ribo-Seq protocols; at present, most Ribo-Seq 43 experiments on bacteria target a size range of 15 to 45 nucleotides, as was used by Li et al, 44 2014 1 . Hence, bacterial ribosome profiling protocols must adopt less stringent size selection to 45 comprehensively...

A global data-driven census ofSalmonellasmall proteins and their potential functions in bacterial virulence

“…This notwithstanding, Ribo-seq may yield false-positive sORF candidates, e.g. due to enrichment of abundant transcripts that are protected from degradation or non-coding ribosome-binding transcripts (Fremin and Bhatt 2020 ). Moreover, due to technical limitations, bacterial Ribo-seq data lack a 3-nucleotide periodicity in ORF coverage, which can hamper sORF annotation.…”