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Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation. [Display omitted] • Hfq and small RNAs target mRNAs during transcription before the mRNA folds • Co-transcriptional targeting by Hfq and small RNA is faster and more efficient • Transient Hfq binding poises the small RNA to base pair with complementary sites • Successful targeting often occurs close to the RNA polymerase exit channel Rodgers et al. use single-molecule fluorescence microscopy to study how bacterial small RNA-Hfq complexes find complementary sites during transcription of a target mRNA. The results reveal that small RNA-Hfq complexes recognize RNA sequences close to the RNA polymerase elongation complex, making targeting more efficient during transcription than after transcription. [ABSTRACT FROM AUTHOR]

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes. [Display omitted] • Cryo-EM structures of PNPase in complex with the RNA chaperone Hfq and regulatory RNA • Structural insights into regulatory RNA recognition by Hfq and PNPase • Model for stabilization of regulatory RNAs and facilitation of their functions The conserved exoribonuclease PNPase contributes to RNA turnover in many organisms, but in bacteria the enzyme can be re-programmed by the RNA chaperone Hfq and regulatory RNA to switch from degradative to chaperoning roles in RNA-mediated gene regulation. Dendooven et al. provide structural insight into the basis for this functional switch and details of the recognition of complex regulatory RNAs by Hfq and PNPase. [ABSTRACT FROM AUTHOR]

Bacterial small RNAs (sRNAs) regulate the expression of hundreds of transcripts via base pairing mediated by the Hfq chaperone protein. sRNAs and the mRNA sites they target are heterogeneous in sequence, length, and secondary structure. To understand how Hfq can flexibly match diverse sRNA and mRNA pairs, we developed a single-molecule Förster resonance energy transfer (smFRET) platform that visualizes the target search on timescales relevant in cells. Here we show that unfolding of target secondary structure on Hfq creates a kinetic energy barrier that determines whether target recognition succeeds or aborts before a stable anti-sense complex is achieved. Premature dissociation of the sRNA can be alleviated by strong RNA-Hfq interactions, explaining why sRNAs have different target recognition profiles. We propose that the diverse sequences and structures of Hfq substrates create an additional layer of information that tunes the efficiency and selectivity of non-coding RNA regulation in bacteria. [Display omitted] • Single-molecule microscopy tracks how Hfq pairs small RNAs with mRNA targets • Hfq chaperone unfolds mRNA structure during base pairing with a small RNA • Unfolding creates a kinetic energy barrier that can cause base pairing to abort • Strong RNA-Hfq interactions increase complex stability and curtail failed attempts Małecka and Woodson visualize how bacterial small RNAs and Hfq bind mRNAs with single-molecule resolution. Structured targets are unfolded by Hfq during sRNA base pairing but increase the probability that the process will abort. These failures help explain why certain targets are regulated more efficiently than others during a stress response. [ABSTRACT FROM AUTHOR]

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli , we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs. • RNA-RNA interactomes for RNA chaperones ProQ and Hfq are identified by RIL-seq • A significant fraction of the RNA-RNA pairs on ProQ also are found on Hfq • sRNA-mediated regulation is impacted by growth conditions • ProQ blocks RNase III- and Hfq-mediated downregulation by a novel sRNA sponge Melamed and Adams et al. compare the RNA-RNA interactomes of the well-characterized Hfq and the understudied ProQ RNA chaperones. They examine overlapping RNA pairs bound by both proteins, identify a new RNA sponge for the RybB small RNA, and document that ProQ can block Hfq activity. [ABSTRACT FROM AUTHOR]