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RNA-based recognition and targeting: sowing the seeds of specificity

Key Points

  • RNAs involved in gene regulation and genome defence need to identify their correct targets in the cell against a background of diverse RNA sequences.

  • Several RNA-based systems have converged on a similar strategy to find their targets. They initially present only a small subregion of the RNA, called a 'seed sequence', to interrogate and specifically bind to their corresponding nucleic acid targets.

  • Argonaute-bound guides present their seed sequence in a pre-organized A-form helix that is already in the correct configuration to bind the mRNA target.

  • Cas9–CRISPR RNA (crRNA) also presents the crRNA seed region in an A-form helix to interrogate potential DNA targets.

  • The Hfq-bound small RNA RydC presents its seed sequence in an extended configuration at the site in Hfq that is involved in its annealing activity.

  • Two general principles have emerged: first, the seed sequence is presented in a conformation that facilitates the search for, and interaction with, target nucleic acids and second, target recognition and conformational changes are coupled within the ribonucleoprotein to ensure that the correct RNA or DNA is regulated.

Abstract

RNA is involved in the regulation of multiple cellular processes, often by forming sequence-specific base pairs with cellular RNA or DNA targets that must be identified among the large number of nucleic acids in a cell. Several RNA-based regulatory systems in eukaryotes, bacteria and archaea, including microRNAs (miRNAs), small interfering RNAs (siRNAs), CRISPR RNAs (crRNAs) and small RNAs (sRNAs) that are dependent on the RNA chaperone protein Hfq, achieve specificity using similar strategies. Central to their function is the presentation of short 'seed sequences' within a ribonucleoprotein complex to facilitate the search for and recognition of targets.

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Figure 1: The biogenesis, loading and targeting of RNA in distinct RNA-based regulatory systems.
Figure 2: Presentation of seed sequences in miRNA and siRNA.
Figure 3: crRNA seed presentation.
Figure 4: Hfq-dependent sRNA seed presentation.
Figure 5: Common principles of the target-search process.

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Acknowledgements

The authors are grateful to B. Luisi, A. Eulalio, G. Wagner and members of the authors' laboratories for discussions and comments on the manuscript. The authors also thank S. Geibel for help with the figures.

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Correspondence to Jörg Vogel or Jennifer A. Doudna.

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Competing interests

J.A.D. is executive director of the Innovative Genomics Initiative at the University of California, Berkeley (UC Berkeley) and the University of California, San Francisco (UCSF). J.A.D. is a co-founder of Editas Medicine, Intellia Therapeutics and Caribou Biosciences and a scientific adviser to Caribou, Intellia, eFFECTOR Therapeutics and Driver. Funding has been received from the Howard Hughes Medical Institute (HHMI), the US National Institutes of Health, the US National Science Foundation, Roche, Pfizer, the Paul Allen Institute and the Keck Foundation. J.A.D. is employed by HHMI and works at the UC Berkeley. UC Berkeley and HHMI have patents pending for CRISPR technologies on which she is an inventor.

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DATABASES

RCSB Protein Data Bank

Glossary

Loop–loop kissing interactions

Watson–Crick base pairing between the loop nucleotides of two RNA stem loops.

Adaptive immunity

A specific response to an infection by a pathogen based on prior exposure to that pathogen.

RNA-induced silencing complex

(RISC). A ribonucleoprotein complex of an Argonaute protein and an RNA.

Scissile bond

A covalent bond that can be broken by an enzymatic reaction.

PAZ domain

(PIWI–Argonaute–Zwille domain). A domain present in Argonaute proteins that is involved in binding to the 3′ end of the guide.

PIWI domain

(P-element-induced wimpy testis domain). A domain present in Argonaute proteins that contains an RNase H-like active site.

A-form helix

A secondary structure motif found in RNA in which bases are tilted with respect to the helix axis.

Entropy penalty

The thermodynamic cost associated with a loss of conformational entropy on the immobilization of a molecule in a fixed configuration.

High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation

(HITS-CLIP). A sequencing method based on the ultraviolet crosslinking of RNA–protein complexes that is used to identify RNA ligands of RNA-binding proteins.

Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation

(PAR-CLIP). A sequencing method that uses photoactivatable ribonucleosides to crosslink RNA and protein to identify RNAs associated with a particular RNA-binding protein.

Crosslinking, ligation and sequencing of hybrids

(CLASH). A sequencing method used to identify RNAs and their targets by ligating them together when they are bound to a specific RNA-binding protein.

HNH domain

A nuclease domain within Cas9 that is related to McrA-like restriction endonucleases.

RuvC domain

A nuclease domain within Cas9 that is related to the RuvC endonuclease that cuts Holliday junctions during homologous recombination.

Total internal reflection microscopy

(TIRFM).A microscopy technique that uses an evanescent wave to specifically excite fluorophore-labelled molecules close to a surface.

Sm/Lsm superfamily

A large family of proteins present in all three domains of life and involved in RNA processing and degradation.

Shine–Dalgarno sequence

A 5–10-nucleotide sequence upstream of the initiation codon involved in defining where bacterial translation initiates.

Chemical footprinting

Methods used to map RNA secondary and tertiary structures based on the accessibility of nucleotides to specific chemicals.

Small-angle X-ray scattering

(SAXS). An X-ray scattering method that provides information on the size and shape of biological molecules in solution.

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Gorski, S., Vogel, J. & Doudna, J. RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol 18, 215–228 (2017). https://doi.org/10.1038/nrm.2016.174

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