Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation.

By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3'UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

Unusual C΄/D΄ motifs enable box C/D snoRNPs to modify multiple sites in the same rRNA target region.

Eukaryotic box C/D small nucleolar (sno)RNPs catalyse the site-specific 2΄-O-methylation of ribosomal RNA. The RNA component (snoRNA) contains guide regions that base-pair with the target site to select the single nucleotide to be modified. The terminal C/D and internal C΄/D΄ motifs in the snoRNA, adjacent to the guide region, function as binding sites for the snoRNP proteins including the enzymatic subunit fibrillarin/Nop1. Four yeast snoRNAs are unusual in that they are predicted to methylate two nucleotides in a single target region. In each case, the internal C΄/D΄ motifs from these snoRNAs differ from the consensus. Our data indicate that the C΄/D΄ motifs in snR13, snR48 and U18 form two alternative structures that lead to differences in the position of the proteins bound to this motif. We propose that each snoRNA forms two different snoRNPs, subtly different in how the proteins are bound to the C΄/D΄ motif, leading to 2΄-O-methylation of different nucleotides in the target region. For snR48 and U18, the unusual C΄/D΄ alone is enough for the modification of two nucleotides. However, for the snR13 snoRNA the unusual C΄/D΄ motif and extra base-pairing, which stimulates rRNA 2΄-O-methylation, are both critical for multiple modifications in the target region.

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing.

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.