Tan Inoue

Design and development of a catalytic ribonucleoprotein

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA‐binding protein were designed and constructed based upon high‐resolution structures of the corresponding prototype molecules, the Tetrahymena group I self‐splicing intron RNA and two proteins (bacteriophage λN and HIV Rev proteins) containing RNA‐binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.

Design, Construction, and Analysis of a Novel Class of Self-Folding RNA

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.

Coordinated control of a designed trans-acting ligase ribozyme by a loop–receptor interaction

We previously developed a synthetic cis‐acting RNA ligase ribozyme with 3′–5′ joining activity termed “DSL” (designed and selected ligase). DSL was easily transformed into a trans‐acting form because of its highly modular architecture. In this study, we investigated the modular properties and turnover capabilities of a trans‐acting DSL, tDSL‐1/GUAA. tDSL‐1/GUAA exhibited remarkably high activity compared with the parental cis‐acting DSL, and it attained a high turnover number. Taken together, the results indicate that a loop–receptor interaction plays a significant role in determining the activity of the trans‐acting ribozyme and in its ability to perform multiple turnovers of the reaction.

The role of peptide motifs in the evolution of a protein network

Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we have constructed an artificial protein library by mixing four peptide motifs shared among the Bcl-2 family proteins that positively or negatively regulate the apoptosis networks. We found one strong pro-apoptotic protein, d29, and two proteins having moderate, but unambiguous anti-apoptotic functions, a10 and d16, from the 28 tested clones. Thus both the pro- and anti-apoptotic modulators were present in the library, demonstrating that functional proteins with opposing effects can emerge from a single pool prepared from common motifs. Motif programming studies have exhibited that the annotated function of the motifs were significantly influenced by the context that the motifs embedded. The results further revealed that reshuffling of a set of motifs realized the promiscuous state of protein, from which disparate functions could emerge. Our finding suggests that motifs contributed to the plastic evolvability of the protein network.

Cloning and characterization of novel extensin-like cDNAs that are expressed during late somatic cell phase in the green alga Volvox carteri.

Asexual individuals of the green alga Volvox carteri consist of two cell types, somatic and reproductive cells. The somatic cells are terminally differentiated post-mitotic cells which undergo gradual senescence leading to cell death in every generation. To understand the gene expression programs associated with senescence of somatic cells, we cloned two cDNAs, LSG1 and LSG2, that are preferentially expressed during this late developmental stage. These two cDNAs were deduced to encode Pro-rich motifs characteristic of extensin proteins that are components of the extracellular matrix. LSG1 also resembled genes encoding plant pathogenesis-related protein 1 (PR-1), while LSG2 showed similarities with genes encoding matrix metalloproteinases, including a gamete lytic enzyme of Chlamydomonas. We also found that S9, one of the late somatic cDNAs previously cloned by Tam and Kirk (Dev. Biol. 145 (1991) 51), was deduced to encode a protein with a composition similar to LSG2. The expression of PR-1 and a matrix-metalloproteinase-encoding gene has been shown to be induced during senescence in higher plants. These results indicate that some of the late somatic genes in V. carteri are related to the senescence-associated genes in higher plants.

Rational optimization of the DSL ligase ribozyme with GNRA/receptor interacting modules

The DSL ribozyme is a class of artificial ligase ribozymes with a highly modular architecture, which catalyzes template-directed RNA ligation on a helical substrate module that can be either covalently connected (cis-DSL) or physically separated (trans-DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively. In this study, we have rationally designed and analyzed the catalytic and self-assembly properties of several trans-DSL ribozymes with different sets of natural and artificial GNRA-receptor clamps. Two variants newly designed in this study showed significantly enhanced catalytic properties with respect of the original trans-DSL construct. While this work allows dissection of the turnover and catalytic properties of the trans-DSL ribozyme, it also emphasizes the remarkable modularity of RNA tertiary structure for nano-construction of complex functions.

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron.

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3 terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a Bulge-and-Twist (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

Redesign of an artificial ligase ribozyme based on the analysis of its structural elements.

The catalytic and folding properties of “DSL ribozyme” were investigated. Thisartificial ligase ribozyme was constructed by installing a catalytic unit to a designedself-folding RNA. The self-folding RNA was composed of three helices connected viatwo tertiary interactions that served as scaffolding in the molecular design. The presentanalysis revealed that the tertiary interaction between the GAAA loop and its specificreceptor plays a crucial role in the folding of the active structure and the precisepositioning of the catalytic site. On the basis of the analyses, the ribozyme wasredesigned and converted to two advanced forms -- a smaller derivative with appreciablecatalytic activity and a derivative with RNA polymerase-like activity. The studydemonstrates that redesign of an artificial ribozyme is effective and efficient if itsstructural elements are finely resolved. This kind of molecular transformation shouldserve as a prototypic model for understanding the molecular organization and evolutionof naturally occurring ribozymes.

Artificial Modules for Enhancing Rate Constants of a Group I Intron Ribozyme without a P4-P6 Core Element

In this paper we report newly selected artificial modules that enhance the kcat values comparable with or higher than those of the wild-type ribozyme with broad substrate specificity. The elements required for the catalysis of Group I intron ribozymes are concentrated in the P3-P7 domain of their core region, which consists of two conserved helical domains, P4-P6 and P3-P7. Previously, we reported the in vitro selection of artificial modules residing at the peripheral region of a mutant Group I ribozyme lacking P4-P6. We found that derivatives of the ribozyme containing the modules performed the reversal of the first step of the self-splicing reaction efficiently by using their affinity to the substrate RNA, although their kcat values and substrate specificity were uninfluenced and limited, respectively. The results show that it is possible to add a variety of new domains at the peripheral region that play a role comparable with that of the conserved P4-P6 domain.

Self-splicing of the Tetrahymena group I ribozyme without conserved base-triples

Group I introns share a conserved core region consisting of two domains, P8‐P3‐P7 and P4‐P6, joined by four base‐triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4‐P6 and the conserved base‐triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions.