The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.     

The double-stranded RNA-binding motif, a versatile macromolecular docking platform

The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.     

The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I

Pseudouridine synthases catalyze the isomerization of specific uridines to pseudouridine in a variety of RNAs, yet the basis for recognition of the RNA sites or how they catalyze this reaction is unknown. The crystal structure of pseudouridine synthase I from Escherichia coli, which, for example, modifies positions 38, 39 and/or 40 in tRNA, reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein. Each cleft contains a highly conserved aspartic acid located at its center. The structural domains have a topological similarity to those of other RNA-binding proteins, though the mode of interaction with tRNA appears to be unique. The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation.     

Design of RNA-binding proteins and ligands

The rapidly expanding database of RNA structures and protein complexes is beginning to lead to the successful design of specific RNA-binding molecules. Recent combinatorial and structure-based approaches have utilized known nucleic-acid-binding scaffolds from both proteins and small molecules to display a relatively small set of functional groups often used in protein--RNA recognition. Several studies have shown that the tethering of multiple binding modules can enhance RNA-binding affinity and specificity, a strategy also commonly used in DNA recognition.     

The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence.

The yeast and mammalian branchpoint sequence binding proteins (BBP and mBBP/SF1) contain both KH domain and Zn knuckle RNA-binding motifs. The single KH domain of these proteins is sufficient for specific recognition of the pre-mRNA branchpoint sequence (BPS). However, an interaction is only apparent if one or more accessory modules are present to increase binding affinity. The Zn knuckles of BBP/mBBP can be replaced by an RNA-binding peptide derived from the HIV-1 nucleocapsid protein or by an arginine-serine (RS)7 peptide, without loss of specificity. Only the seven-nucleotide branchpoint sequence and two nucleotides to either side are necessary for RNA binding to the chimeric proteins. Therefore, we propose that all three of these accessory RNA-binding modules bind the phosphate backbone, whereas the KH domain interacts specifically with the bases of the BPS. Proteins and protein complexes with multiple RNA-binding motifs are frequent, suggesting that an intimate collaboration between two or more motifs will be a general theme in RNA-protein interactions.     

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.     

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.     

RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.     

RNA recognition by the DNA end-binding Ku heterodimer

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem–loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem–loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.     

The HicAB cassette, a putative novel, RNA-targeting toxin-antitoxin system in archaea and bacteria

Toxin-antitoxin systems (TAS) are abundant, diverse, horizontally mobile gene modules that encode powerful resistance mechanisms in prokaryotes. We use the comparative-genomic approach to predict a new TAS that consists of a two-gene cassette encoding uncharacterized HicA and HicB proteins. Numerous bacterial and archaeal genomes encode from one to eight HicAB modules which appear to be highly prone to horizontal gene transfer. The HicB protein (COG1598/COG4226) has a partially degraded RNAse H fold, whereas HicA (COG1724) contains a double-stranded RNA-binding domain. The stable combination of these two domains suggests a link to RNA metabolism, possibly, via an RNA interference-type mechanism. In most HicB proteins, the RNAse H-like domain is fused to a DNA-binding domain, either of the ribbon-helix-helix or of the helix-turn-helix class; in other TAS, proteins containing these DNA-binding domains function as antitoxins. Thus, the HicAB module is predicted to be a novel TAS whose mechanism involves RNA-binding and, possibly, cleavage.     

mRNPs take shape by CLIPPING and PAIRING

The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations.     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.     

Themes in RNA-protein recognition.

Atomic resolution structures are now available for more than 20 complexes of proteins with specific RNAs. This review examines two main themes that appear in this set of structures. A "groove binder" class of proteins places a protein structure (alpha-helix, 310-helix, beta-ribbon, or irregular loop) in the groove of an RNA helix, recognizing both the specific sequence of bases and the shape or dimensions of the groove, which are sometimes distorted from the normal A-form. A second class of proteins uses beta-sheet surfaces to create pockets that examine single-stranded RNA bases. Some of these proteins recognize completely unstructured RNA, and in others RNA secondary structure indirectly promotes binding by constraining bases in an appropriate orientation. Thermodynamic studies have shown that binding specificity is generally a function of several factors, including base-specific hydrogen bonds, non-polar contacts, and mutual accommodation of the protein and RNA-binding surfaces. The recognition strategies and structural frameworks used by RNA binding proteins are not exotically different from those employed by DNA-binding proteins, suggesting that the two kinds of nucleic acid-binding proteins have not evolved independently.     

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.     

Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P.

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.