Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field.     

Hairpin RNA: a secondary structure of primary importance

An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought. Received 21 November 2005; received after revision 3 January 2006; accepted 11 January 2006     

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5′-hydroxyl group and a 2′,3′-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed. Received 21 June 2001; received after revision 18 July 2001; accepted 20 July 2001     

RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence

The double-stranded RNA binding domain (dsRBD) is a small protein domain of 65–70 amino acids adopting an αβββα fold, whose central property is to bind to double-stranded RNA (dsRNA). This domain is present in proteins implicated in many aspects of cellular life, including antiviral response, RNA editing, RNA processing, RNA transport and, last but not least, RNA silencing. Even though proteins containing dsRBDs can bind to very specific dsRNA targets in vivo, the binding of dsRBDs to dsRNA is commonly believed to be shape-dependent rather than sequence-specific. Interestingly, recent structural information on dsRNA recognition by dsRBDs opens the possibility that this domain performs a direct readout of RNA sequence in the minor groove, allowing a global reconsideration of the principles describing dsRNA recognition by dsRBDs. We review in this article the current structural and molecular knowledge on dsRBDs, emphasizing the intricate relationship between the amino acid sequence, the structure of the domain and its RNA recognition capacity. We especially focus on the molecular determinants of dsRNA recognition and describe how sequence discrimination can be achieved by this type of domain.     

Membrane protein folding on the example of outer membrane protein A of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded -barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the -barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of -sheet secondary structure and -barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of -barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.     

RNA二级结构的2D图形表示及相似性分析

结合RNA序列的核苷酸分类提出了一种新的RNA二级结构的2D图形表示法。该图形表示法能够唯一地表示RNA二级结构,而且图形是无退化的,即不存在重叠或交叉现象。把该图形表示法应用于9个病毒RNA二级结构,通过计算这些序列的数字特征和序列之间的相似性,实验表明该图形表示法是可行的。     

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.     

RNA processing and human disease

Gene expression involves multiple regulated steps leading from gene to active protein. Many of these steps involve some aspect of RNA processing. Diseases caused by mutations that directly affect RNA processing are relatively rare compared with mutations that disrupt protein function. The vast majority of diseases of RNA processing result from loss of function of a single gene due to mutations in cis-acting elements required for pre-messenger RNA (mRNA) splicing. However, a few diseases are caused by alterations in the trans-acting factors required for RNA processing and in the vast majority of cases it is the pre-mRNA splicing machinery that is affected. Clearly, alterations that disrupt splicing of pre-mRNAs from large numbers of genes would be lethal at the cellular level. A common theme among these diseases is that only subsets of genes are affected. This is consistent with an emerging view that different subsets of exons require different sets of cis-acting elements and trans-acting factors.     

Secondary and topographic structure of ribosomal RNA 16S of Escherichia coli

We present a model for the secondary structure of 16S ribosomal RNA from E. coli. This model has been deduced by restricting the total number of theoretical base pairings using the following criteria: (1) susceptibility of residues towards enzymatic probes that are specific for either paired or single stranded regions; (2) reactivity of certain residues to chemical modification; (3) evidence for medium and long range interactions; (4) comparative analysis of ribosomal RNA sequences from other organisms.     

Hepatitis C viral RNA: challenges and promises

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus of the Flaviviridae family, is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Its RNA is difficult to study because biological materials are scarce and RNA replication is of low efficiency. This review focuses on the structure and functions of HCV RNA along with their biological and clinical significance. Despite the challenging characteristics of HCV, significant progress has been made in understanding the properties of HCV RNA and developing viral replication systems toward the improvement of antiviral therapies. Received 15 January 2001; received after revision 2 March 2001; accepted 18 April 2001     

Amyloid fibrils from the viewpoint of protein folding

In amyloid related diseases, proteins form fibrillar aggregates with highly ordered -sheet structure regardless of their native conformations. Formation of such amyloid fibrils can be reproducible in vitro using isolated proteins/peptides, suggesting that amyloid fibril formation takes place as a result of protein conformational change. In vitro studies revealed that perturbation of the native structure is important for the fibril formation, and it is suggested that the mechanisms of amyloid fibril formation share the mechanisms of protein folding. In particular, amyloid fibril formation is similar to one of the common features of proteins, i.e. amorphous aggregation upon partial unfolding, which is likely driven by hydrophobic interactions through exposed protein interior. However, these molecular associations are distinct phenomena, and identifying factors that lead to amyloid fibril formation would precede our understanding of the mechanisms of amyloid fibrillization. The necessity of understanding the nature of protein denatured states is also suggested.Received 6 July 2003; accepted 19 August 2003     

RNA uridylyltransferases

The terminal RNA uridylyltransferases (TUTases) catalyze transfer of UMP residues to the 3' hydroxyl group of RNA. These activities are widespread among eukaryotes and appear to be involved in a variety of RNA-processing pathways. Recent studies of RNA editing in trypanosomatids have provided the first insights into the biological functions of RNA uridylyltransferases, which had eluded biochemical identification despite 30-year-old evidence of such activities in mammals and plants. Comparative sequence analysis of trypanosomal TUTases and their homologs revealed by large-scale genomic projects demonstrates a significant level of biochemical and structural diversity between putative uridylyltransferases. The conserved catalytic domain has acquired additional protein modules and appears to have adapted to perform functionally distinct tasks of guided U-insertion into mRNA and constrained addition of an oligo[U] tail to guide RNAs. Here I discuss the current knowledge of this novel enzyme family and possible roles of RNA uridylylation in the regulation of gene expression.     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast

We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome. Many stretches of sequence are present which are homologous to the E. coli 16S ribosomal RNA gene. The gene sequence can be folded into a secondary structure according to the [1] model on bacterial ribosomal RNAs. The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions. In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome.     

Biogenesis of prokaryotic pores.

The prokaryotic pore-forming proteins are synthesized in the cytoplasm, and are assembled in their functional form in the outer membrane. They begin to traverse the cytoplasmic membrane via the SecY/SecA export pathway, which is shared also by periplasmic proteins. The sorting signals that direct these proteins to the outer membrane could be present in the three-dimensional conformations of the proteins, but some results suggest that they may be present in short, contiguous sequences. Outer membrane proteins share a rather hydrophilic amino acid composition, and appear to be rich in beta-sheets (with the exception of lipoproteins). This observation as well as the demonstration of periplasmic export intermediates favor the secretion pathway through the periplasm, as opposed to export through fusion sites between the inner and the outer membrane, but such intermediates have not yet been observed with the wild type proteins under physiological conditions.     

Re-creating an RNA world

The RNA world hypothesis states that life originated via a system based on RNA genomes and RNA catalysts. Researchers have been trying to develop such a system since catalytic RNAs (ribozymes) were discovered in 1982. This review summarizes the recent progress made in that endeavor and outlines the obstacles that remain to be overcome. After giving a short background on prebiotic chemistry and in vitro evolution, the discussion focuses on the generation of three important components of an RNA world: a sufficient polymerase ribozyme, self-replicating membrane compartments and ribozymes that are capable of performing basic metabolic processes. Received 31 January 2006; accepted 15 March 2006     

The folding process of hen lysozyme: a perspective from the ‘new view’

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the ‘new view’ of protein folding based on energy surfaces and land scapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.