Gabriele Varani

RNA-binding proteins: modular design for efficient function

Many RNA-binding proteins have modular structures and are composed of multiple repeats of just a few basic domains that are arranged in various ways to satisfy their diverse functional requirements. Recent studies have investigated how different modules cooperate in regulating the RNA-binding specificity and the biological activity of these proteins. They have also investigated how multiple modules cooperate with enzymatic domains to regulate the catalytic activity of enzymes that act on RNA. These studies have shown how, for many RNA-binding proteins, multiple modules define the fundamental structural unit that is responsible for biological function.

RNA recognition by a Staufen double-stranded RNA-binding domain

The double‐stranded RNA‐binding domain (dsRBD) is a common RNA‐binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA‐binding activity of dsRBD3 is required in vivo for Staufen‐dependent localization of bicoid and oskar mRNAs. Using high‐resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A‐form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single‐stranded loop that caps the RNA helix. Interactions between helix α1 and single‐stranded RNA may be important determinants of the specificity of dsRBD proteins.

The structure and function of small nucleolar ribonucleoproteins

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequence-specific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose-2′-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs.

The G·U wobble base pair: A fundamental building block of RNA structure crucial to RNA function in diverse biological systems

The G·U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson–Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G·C or A·U base pairs. The G·U wobble base pair also has unique chemical, structural, dynamic and ligand‐binding properties, which can only be partially mimicked by Watson–Crick base pairs or other mispairs. These features mark sites containing G·U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.

RNA structure and NMR spectroscopy

RNA molecules perform a wide variety of biological functions, from enzymic activity to storage and propagation of genetic information.

Protein families and RNA recognition

This minireview series examines the structural principles underlying the biological function of RNA‐binding proteins. The structural work of the last decade has elucidated the structures of essentially all the major RNA‐binding protein families; it has also demonstrated how RNA recognition takes place. The ribosome structures have further integrated this knowledge into principles for the assembly of complex ribonucleoproteins. Structural and biochemical work has revealed unexpectedly that several RNA‐binding proteins bind to other proteins in addition to RNA or instead of RNA. This tremendous increase in the structural knowledge has expanded not only our understanding of the RNA recognition principle, but has also provided new insight into the biological function of these proteins and has helped to design better experiments to understand their biological roles.

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP‐RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The β‐sheet of the domain provides an extensive RNA‐binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single‐stranded nucleotides and residues on the β‐sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA‐protein recognition, much closer to our current understanding of protein‐protein recognition than that of DNA‐protein recognition.

Recent advances in RNA-protein recognition

The past few years have witnessed remarkable progress in knowledge of the structure and function of RNA-binding proteins and their RNA complexes. X-ray crystallography and NMR spectroscopy have provided structures for all major classes of RNA-binding proteins, both alone and complexed with RNA. New computational and experimental tools have provided unprecedented insight into the molecular basis of RNA recognition.

The NMR structure of the 38 kDa U1A protein - PIE RNA complex reveals the basis of cooperativity in regulation of polyadenylation by human U1A protein.

The status of the poly(A) tail at the 3′-end of mRNAs controls the expression of numerous genes in response to developmental and extracellular signals. Poly(A) tail regulation requires cooperative binding of two human U1A proteins to an RNA regulatory region called the polyadenylation inhibition element (PIE). When bound to PIE RNA, U1A proteins also bind to the enzyme responsible for formation of the mature 3′-end of most eukaryotic mRNAs, poly(A) polymerase (PAP). The NMR structure of the 38 kDa complex formed between two U1A molecules and PIE RNA shows that binding cooperativity depends on helix C located at the end of the RNA-binding domain and just adjacent to the PAP-interacting domain of U1A. Since helix C undergoes a conformational change upon RNA binding, the structure shows that binding cooperativity and interactions with PAP occur only when U1A is bound to its cognate RNA. This mechanism ensures that the activity of PAP enzyme, which is essential to the cell, is only down regulated when U1A is bound to the U1A mRNA.

Eukaryotic translation initiation: there are (at least) two sides to every story

The eukaryotic cap and poly(A) tail binding proteins, eIF4E and Pab1p, play important roles in the initiation of protein synthesis. The recent structures of the complex of eIF4E bound to the methylated guanosine (cap) found at the 5′end of messenger RNA (mRNA), the complex of eIF4E bound to peptide fragments of two related translation factors (eIF4G and 4E-BP1), and the complex of the N-terminal fragment of Pab1p bound to polyadenylate RNA have revealed that eIF4E and Pab1p contain at least two distinct functional surfaces. One surface is used for binding mRNA, and the other for binding proteins involved in translation initiation.