Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.

Summary: Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one the largest ES, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L increase amino acid misincorporation, as well as read-through and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.

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RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms.

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.urinary tract infection | fimS | type 1 pili | phase variation | saturating chromosomal mutagenesis U rinary tract infections (UTIs) are common infections that mostly affect women with an annual cost of billions of dollars (1). They are caused mainly by uropathogenic Escherichia coli (UPEC), which rely on several virulence factors to cause disease (2); type 1 pili (T1P) are perhaps the most important of these (3). T1P present a tip adhesin, FimH, that binds specifically to mannose, which is found on several glycosylated surface proteins, such as uroplakins and α1-and β3-integrins (4, 5), in the mammalian bladder. Binding to mannosylated proteins initiates a cascade of events including UPEC invasion of host tissues (2), formation of intracellular structures (6), and activation of the host immune response (7). The interplay among these events determines the outcome of infection (8), ranging from full clearance to chronic active cystitis. T1P are important for most of these UTI stages and demonstrate dynamic regulation of expression that can be seen directly in human infections and urine (9, 10).Thus, understanding T1P regulation is fundamental to understanding UTI. T1P have been well studied (11), mostly in laboratory-adapted E. coli strains. T1P are encoded by the fim operon; their expression is phase variable via inversion of fimS, a chromosomal DNA segment containing the fim promoter (12). This binary epigenetic switch results in bacteria switching between fimbriated and nonfimbriated states. Inversion is mediated by the FimB and FimE recombinases, whose genes are located immediately upstream of fimS (13) and are found in most E. coli strains (14). Inversion is influenced by numerous signals, such as temperatu...

The hundreds of copies of ribosomal-DNA genes are the dark-matter of the human genome as it is unknown whether they possess sequence variation that forms different types of ribosomes. Here, we have overcome the technical hurdle of long-read sequencing of full-length ribosomal-RNA (rRNA) and developed an efficient algorithm for rRNA-variant detection. We discovered hundreds of variants that are not silent but are incorporated into translating ribosomes. These include tens of abundant variants within functionally important domains of the ribosome. Strikingly, variants assemble into distinct ribosome subtypes encoded on different chromosomes. With this first atlas of expressed rRNA-variants, we discover the impact of rRNA variation on health and disease. Across human tissues, we observe tissue-specific variant expression in endoderm/ectoderm derived tissues. In cancer, low abundant rRNA-variants become highly expressed. Together, this study provides a curated atlas for exploring rRNA variation and functionally links ribosome variation to tissue-specific biology and cancer.

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