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Structural Mechanisms of RNA Recognition: Sequence-Specific and Non-Specific RNA-Binding Proteins and the Cas9-RNA-DNA Complex

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Structural Mechanisms of RNA Recognition: Sequence-Specific and Non-Specific RNA-Binding Proteins and the Cas9-RNA-DNA Complex Cell. Mol. Life Sci. (2015) 72:1045–1058 DOI 10.1007/s00018-014-1779-9 Cellular and Molecular Life Sciences REVIEW Structural mechanisms of RNA recognition: sequence-specific and non-specific RNA-binding proteins and the Cas9-RNA-DNA complex Ting Ban • Jian-Kang Zhu • Karsten Melcher • H. Eric Xu Received: 26 June 2014 / Revised: 28 October 2014 / Accepted: 10 November 2014 / Published online: 29 November 2014 Ó Springer Basel 2014 Abstract RNA-binding proteins play crucial roles in RNA specificity. We also highlight the structural mechanism of processing and function as regulators of gene expression. sequence-dependent and -independent interactions in the Recent studies have defined the structural basis for RNA Cas9-RNA-DNA complex. recognition by diverse RNA-binding motifs. While many RNA-binding proteins recognize RNA sequence non-spe- Keywords RNA recognition Á RNA-binding protein Á cifically by associating with 50 or 30 RNA ends, sequence- Tandem repeat module Á Sequence-specific recognition Á specific recognition by RNA-binding proteins is typically Protein–protein interaction achieved by combining multiple modular domains to form complex binding surfaces. In this review, we present Abbreviations examples of structures from different classes of RNA-bind- PPR Pentatricopeptide repeat ing proteins, identify the mechanisms utilized by them to Pre-mRNA Precursor messenger RNA target specific RNAs, and describe structural principles of RBDs RNA-binding domains how protein–protein interactions affect RNA recognition TPR Tetratricopeptide repeat PUF Pumilio-FBF T. Ban Á H. E. Xu TZF TTP-like zinc finger VARI-SIMM Center, Center for Structure and Function of Drug AGO Argonaute-like Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, FBF fem-3 binding factor Shanghai 201203, China ssRNA Single-stranded RNA TTP Tristetraprolin T. Ban Á J.-K. Zhu PIWI P-element-induced wimpy testes Shanghai Center for Plant Stress Biology and Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute of PAZ PIWI, Argonaute, and Zwille Biological Sciences, Chinese Academy of Sciences, 300 Fenglin MDA5 Melanoma differentiation-associated gene 5 Road, Shanghai 200032, China RIG-I Retinoic acid-inducible gene I PPP-RNA 50 triphosphate RNA J.-K. Zhu Department of Horticulture and Landscape Architecture, Purdue RRM RNA recognition motif University, West Lafayette, IN 47906, USA U2AF U2 auxiliary factor hnRNP Heterogeneous nuclear ribonucleoprotein K. Melcher Á H. E. Xu CRISPR Clustered regularly interspaced short Laboratory of Structural Sciences and Laboratory of Structural Biology and Biochemistry, Van Andel Research Institute, 333 palindromic repeats Bostwick Ave., N.E, Grand Rapids, MI 49503, USA Present Address: Introduction T. Ban (&) Roche Innovation Center Shanghai, 720 Cailun Road, Shanghai 201203, People’s Republic of China RNA processing plays critical roles in regulating gene e-mail: [email protected] expression, including pre-mRNA splicing, RNA- 123 1046 T. Ban et al. interference, and mRNA stabilization. RNA-binding pro- structure of apo IFIT5 as well as structures of IFIT5 in teins (RBPs) are central to this form of regulation. They complex with three different RNA oligonucleotides: IFIT5- function in nearly all pathways that are associated with oligo-A, IFIT5-oligo-C and IFIT5-oligo-U. All three RNA processing and promote the activity of functional and complex structures share the exact same oligonucleotide structural RNA molecules [13, 30]. binding pocket. The 50-triphosphate group is buried deeply Most RNA-binding proteins contain multiple RNA- within the pocket and makes a multitude of hydrogen bond binding domains, many of which are structurally and and salt-bridge interactions with residues on helix a2 (E33, functionally modular. Normally, a single modular RNA- T37 and Q41) and the concave inner pocket surface (K150, binding domain (RBD) does not have sufficient binding Y250 and R253) (Fig. 1b). Because critical interactions are capacity to interact with RNA in a sequence-specific made with the c-phosphate group, the pocket is unlikely to manner because the recognition sequences are often too bind 50-monophosphorylated or 50-hydroxylated RNA with short. Instead, multiple RNA-binding domains are tethered considerable affinity. Thus, the structure of the IFIT5 TPR together to create a much larger binding interface that domain has evolved to specifically interact with 50 tri- recognizes a longer sequence to increase specificity and phosphate-containing RNAs, and through this binding enhance the affinity for target RNAs [19, 24]. To under- mechanism to distinguish between host and non-self viral stand the function of RNA-binding proteins, it is important nucleic acids. to know how these domains function together as RNA The second example is the human Argonaute-2 (hAgo2) recognition units. protein. The crystal structure of hAgo2 bound to microR- Structural biology has revealed the molecular basis for NA-20a revealed that the microRNA interacts with all four RNA recognition by individual domains [19]. Here, we domains of hAgo2 as well as with the linkers connecting focus on how RNA-binding proteins recognize their target these domains (Fig. 1c). This structure provides a frame- RNAs, summarize the RNA recognition mechanisms by work for how Ago proteins recognize microRNAs and different types of RBDs and their modular arrangements, function in inducing RNA-directed silencing [12]. The 50- and discuss the importance of dimer formation and pro- end of miR-20a is tethered to hAgo2 through interaction tein–protein interactions in precise RNA recognition. We between the terminal monophosphate of ‘‘U’’ and a binding also review the recent structures of Cas9/RNA complex, pocket composed of the Mid and PIWI domains. K533, the essential component of the new genome editing tech- Y529, K570, K566, and Q545 from the Mid domain as well nology [4, 17, 28]. as R812 from the PIWI domain interact with the mono- phosphate of U1 through salt bridges and hydrogen bonds (Fig. 1d). The following seven nucleotides (50-AAA- Non-sequence specific recognition GUGCU-30) form the seed sequence, which is located in a narrow RNA binding groove. Recognition of this sequence A series of structures of RBPs that recognize selective is mainly mediated through base-independent interactions target RNAs in a non-sequence-specific manner have been with backbone phosphates and sugars, consistent with the reported. Most of these proteins recognize target RNA via high sequence variability of seed sequences and with the binding of marker groups at the 50 or 30 ends of RNA ability of Argonaute proteins to accept many small RNA fragments [24]. For instance, PIWI (P-element induced sequences [37]. The structure of Ago proteins is very wimpy testes) utilizes a highly conserved binding pocket to conserved among eukaryotic and prokaryotic homologs, recognize the defining 50 phosphate group in the siRNA indicating a conserved mode of RNA binding as the basis guide strand [26]; PAZ (PIWI, Argonaute, and Zwille) for 50-nucleotide recognition of the guide strand. recognizes single-stranded 30 overhangs of siRNA through Beside single-stranded RNA recognition, non-sequence stacking interactions and hydrogen bonding [33]. In this specific recognition also involves binding of specific RBPs section, we review two recent complex structures of RNA- to double-stranded RNAs. MDA5 (melanoma differentia- bound RBPs that have revealed the molecular basis of how tion-associated gene 5) and RIG-I (retinoic acid-inducible this non-sequence-specific recognition occurs. gene I) are related cytoplasmic viral RNA receptors in the The first example is the innate immune effector IFIT5, a vertebrate innate immune system. Structures of both pro- tetratricopeptide repeat protein that selectively binds viral teins bound to double-stranded (ds) RNAs expanded our RNA in a sequence-independent manner by recognizing understanding of RNA recognition by RBPs and revealed their characteristic free 50 triphosphate ends [1]. IFIT5 complex strategies for RNA signaling [16, 38]. The crystal contains 24 a-helices, which are connected by short linkers structure of RIG-I revealed binding of the ds RNA end to a and which surround a highly positively charged deep narrow, highly basic channel, in which residues make pocket that is well suited for the accommodation of nucleic predominantly contacts with the sugar-phosphate backbone acids (Fig. 1a). The authors determined the crystal of both strands and recognize the viral ds RNA ends. Most 123 Structural mechanisms of RNA recognition 1047 Fig. 1 RBPs can bind target RNAs in a non-sequence specific colored in red, the PIWI domain is dark blue, the PAZ domain is manner, which is dependent on recognition of marker groups at the 50 magenta and the N terminal domain is light blue. The 50 end of miR- and 30 ends of target RNA molecules. a Overall structural views of 20a is trapped at the interface of the Mid domain and PIWI domain, IFIT5 in absence of RNA presented as cartoon models (PDB and the 30 end of miR-20a is bound to the PAZ domain. RNA is accession code: 4HOQ). b Close-up view of the residues making represented as stick models. d Close-up view of the interactions of the contacts with the RNA 50-triphosphate group (PDB accession code: first miR-20a base (U1) and the terminal monophosphate with hAgo2. 4HOR). The three phosphates are labeled as a, b, c; RNA and the Interacting residues are shown in stick representation with carbons in amino acids that interact with the triphosphate group are presented as pink, nitrogens in blue and oxygens in red. The RNA is shown as stick stick models. The 50 nucleotide (N1) is shown in stick representation, model, with carbons in yellow and phosphors in orange. The Mid with carbon atoms in pink, phosphor atoms in orange, nitrogens in domain is shown in gray and the PIWI domain is dark blue. Hydrogen blue and oxygens in red. c Overall structure of hAgo2 in complex bond and salt bridge interactions are indicated by black dashed lines with miR-20a (PDB accession code: 4F3T).
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