Sumitaka Hasegawa

Apoptosis in neural crest cells by functional loss of APC tumor suppressor gene

Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of β-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where β-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of β-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells.

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

Abstract1. The cellular prion protein, designated PrPc, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrPc plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrPc is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.

An Alternative Transcript Derived from the Trio Locus Encodes a Guanosine Nucleotide Exchange Factor with Mouse Cell-transforming Potential

By screening cDNA expression libraries derived from fresh leukemic cells of adult T-cell leukemia for the potential to transform murine fibroblasts, NIH3T3, we have identified a novel transforming gene, designated Tgat. Expression of Tgat in NIH3T3 resulted in the loss of contact inhibition, increase of saturation density, anchorage-independent growth in a semisolid medium, tumorigenicity in nude mice, and increased invasiveness. Sequence comparison revealed that an alternative RNA splicing of the Trio gene was involved in the generation of Tgat. The Tgat cDNA encoded a protein product consisting of the Rho-guanosine nucleotide exchange factor (GEF) domain of a multifunctional protein, TRIO, and a unique C-terminal 15-amino acid sequence, which were derived from the exons 38-46 of the Trio gene and a novel exon located downstream of its last exon (exon 58), respectively. A Tgat mutant cDNA lacking the C-terminal coding region preserved Rho-GEF activity but lost the transforming potential, indicating an indispensable role of the unique sequence. On the other hand, treatment of Tgat-transformed NIH3T3 cells with Y-27632, a pharmacological inhibitor of Rho-associated kinase, abrogated their transforming phenotypes, suggesting the coinvolvement of Rho-GEF activity. Thus, alternative RNA splicing, resulting in the fusion protein with the Rho-GEF domain and the unique 15 amino acids, is the mechanism generating the novel oncogene, Tgat.

Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells

Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, β-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of β-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.

Impaired Motor Coordination in Mice Lacking Prion Protein

Abstract1. Prion protein (PrPC) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrPC remains to be elucidated.2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrPC plays a role in the long-term survival of Purkinje cells.3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrPC for both the development of prion diseases and the prion propagation.

Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans‐splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme‐mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180‐fold, and by ten‐fold for the stably expressed target. siRNA‐mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme‐based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

Detection of mRNA in mammalian cells with a split ribozyme reporter

The detection of mRNA expression in vivo can reveal essential information about basic biology and disease processes. Current methods primarily involve the use of labeled (with fluorophores or radioactive isotopes) antisense oligomers, based on a one-to-one receptor–ligand type of interaction without robust signal amplification, such as molecular beacons. Given that the copy number of a particular mRNA target per cell is generally ~50–1000, it is challenging to detect such a small number of mRNA in vivo with current methods. Here we introduce a new strategy with a mechanism of signal amplification for sensing target mRNAs in mammalian cells. This strategy utilizes the Tetrahymena group I intron ribozyme as an RNA sensor. Group I introns of the ciliated protozoan Tetrahymena thermophila have been shown to catalyze trans-splicing of mRNA molecules in mammalian cells in which the ribozyme splices an attached 3’ exon to a designated splice site on any chosen target RNA. This property has been variously exploited for ribozyme-mediated repairing of mutant mRNA transcripts. We were interested in exploiting this splicing activity to detect mRNAs in mammalian cells because it can potentially achieve robust signal amplification through enzymatic catalysis. Splitreporter technology based on protein (intein) splicing has been demonstrated with Renilla luciferase and green fluorescent protein in detecting protein–protein interactions. The splicing activity of the Tetrahymena ribozyme may be similarly applied to develop a biosensor for mRNA detection. We previously reported a cis-splicing ribozyme construct, Rz156, in which the Tetrahymena group I intron ribozyme was inserted into the coding sequence of the cDNA of the nonsecreted TEM-1 b-lactamase (Bla). Based on this cis-splicing ribozyme, we devised a split-reporter strategy for the detection of target mRNA in cells (Scheme 1). In this design, Rz156 was split at the L1 loop into a pair of plasmid constructs—TRzL and TRzR—each carrying a part of the coding sequence of the Bla reporter and a fragment of the Tetrahymena ribozyme. Both TRzL and TRzR also carried an antisense sequence complementary to a target mRNA, and a six-nucleotide-long linker

Modulating the splicing activity of Tetrahymena ribozyme via RNA self-assembly

The internal guiding sequence (IGS) is normally located at the 5′ end of trans‐splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans‐splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS‐containing substrate, and the IGS‐lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans‐splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self‐assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.