Structured RNA Contaminants in Bacterial Ribo-Seq

Abstract

Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, Ribo-Seq signal can be observed across RNAs that one would not expect to be bound by ribosomes. For example, Escherichia coli Ribo-Seq libraries also capture reads from most noncoding RNAs (ncRNAs). While some of these ncRNAs may overlap coding regions, this alone does not explain the majority of observed signal across ncRNAs. These fragments of ncRNAs in Ribo-Seq data pass all size selection steps of the Ribo-Seq protocol and survive hours of micrococcal nuclease (MNase) treatment. In this work, we specifically focus on Ribo-Seq signal across ncRNAs and provide evidence to suggest that RNA structure, as opposed to ribosome binding, protects them from degradation and allows them to persist in the Ribo-Seq sequencing library preparation. By inspecting these “contaminant reads” in bacterial Ribo-Seq, we show that data previously disregarded in bacterial Ribo-Seq experiments may, in fact, be used to gain partial information regarding the in vivo secondary structure of ncRNAs. IMPORTANCE Structured ncRNAs are pivotal mediators of bioregulation in bacteria, and their functions are often reliant on their specific structures. Here, we first inspect Ribo-Seq reads across noncoding regions, identifying contaminant reads in these libraries. We observe that contaminant reads in bacterial Ribo-Seq experiments that are often disregarded, in fact, strongly overlap with structured regions of ncRNAs. We then perform several bioinformatic analyses to determine why these contaminant reads may persist in Ribo-Seq libraries. Finally, we highlight some structured RNA contaminants in Ribo-Seq and support the hypothesis that structures in the RNA protect them from MNase digestion. We conclude that researchers should be cautious when interpreting Ribo-Seq signal as coding without considering signal distribution. These findings also may enable us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large scale and in vivo through the reanalysis of existing Ribo-Seq data sets.

ORFik: a comprehensive R toolkit for the analysis of translation

“…This is because not all isolated fragments necessarily originate from regions protected by ribosomes. Instead, these can be the product of other sources such as RNA structure, RNA binding proteins or incomplete digestion [ 33 ]. This filtering of sizes is typically performed first in the lab and then computationally.…”

ORFik: a comprehensive R toolkit for the analysis of translation

“…This is because not all isolated fragments necessarily originate from regions protected by ribosomes. Instead, these can be the product of other sources such as RNA structure, RNA binding proteins or incomplete digestion 34 This filtering of sizes is typically performed first in the lab and then computationally. To identify which read lengths most likely originate from actual ribosome protected footprints (RFPs), ORFik identifies read lengths that display 3 nucleotides (nt) periodicity over protein-coding regions, indicative of ribosome translocation (Figure 1A).…”

“…This is because not all isolated fragments necessarily originate from regions protected by ribosomes. Instead, these can be the product of other sources such as RNA structure, RNA binding proteins or incomplete digestion 34 . This filtering of sizes is typically performed first in the lab and then computationally.…”

Spotlight on alternative frame coding: Two long overlapping genes in Pseudomonas aeruginosa are translated and under purifying selection

“…More recently, mRNA protected by ribosomes after enzymatic degradation has been sequenced using “ribosome profiling” (RiboSeq) ( Ingolia et al., 2009 ), showing evidence of translation for antisense transcripts ( Ardern et al., 2020 ; Zehentner et al., 2020a ). However, artifacts may occur due to structured RNAs ( Fremin and Bhatt, 2020 ). Moreover, proteins produced from such transcripts have been claimed to be predominantly nonfunctional in Mycobacterium tuberculosis ( Smith et al., 2019 ).…”