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Gene discovery by ribozyme and siRNA libraries

Nature Reviews Molecular Cell Biology volume 6, pages 413–422 (2005)Cite this article

Key Points

  • Rapid progress in the sequencing of several genomes has provided abundant sequence information, but the functions of many of the genes remain to be determined. So, a simple and reliable knockdown tool is needed, for example, ribozymes or small interfering RNAs (siRNAs), for the construction of genome-wide libraries.

  • The hammerhead ribozyme can be engineered to optimize its activity in the intracellular environment. The introduction of a library of active ribozymes into cells, and the subsequent screening for phenotypic changes, can lead to the rapid identification of gene function.

  • The application of randomized ribozyme libraries to gene discovery in the field of cancer biology has led to the identification of several new genes that function in tumorigenesis and metastasis. In addition, randomized ribozyme libraries have been used to identify other types of genes, such as genes associated with the viral life cycle, apoptotic pathways, Alzheimer's disease, muscle differentiation and neuronal differentiation.

  • RNA interference (RNAi) technology is also attractive as a knockdown tool for gene discovery. Compared with ribozyme libraries, siRNA libraries have a higher suppressive activity but lower specificity. Therefore, because of the strong specificity of ribozymes and the strong activities of siRNAs, it is clear that the two technologies have important, complementary roles in the identification of new genes and their functions.

  • This article describes the use of catalytic RNAs (or ribozymes) as tools for the identification of gene function, and compares the pros and cons of ribozyme and siRNA libraries in large-scale screening.

Abstract

Catalytic RNAs, also known as ribozymes, can be engineered to optimize their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function. For the determination of gene function, ribozyme technology complements another RNA-based tool that is based on libraries of small interfering RNAs.

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Figure 1: Expression of a hammerhead ribozyme.
Figure 2: Strategy for gene discovery using a library of randomized hairpin or hammerhead ribozymes.
Figure 3: Identification of genes involved in metastasis.

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Author notes

  1. Hideo Akashi and Sahohime Matsumoto: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Tokyo, 113-8656, Japan

    Hideo Akashi, Sahohime Matsumoto & Kazunari Taira

  2. Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, Hongo, Tokyo, 113-8655, Japan

    Sahohime Matsumoto

  3. Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City, 305-8562, Japan

    Kazunari Taira

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  1. Hideo Akashi
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Correspondence to Kazunari Taira.

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DATABASES

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ppan

Glossary

ANTISENSE OLIGONUCLEOTIDES

An oligonucleotide that is complementary to a specific mRNA molecule. The antisense oligonucleotide can hybridize with the target mRNA and prevent gene expression.

HAMMERHEAD RIBOZYME

A small catalytic RNA molecule, the two-dimensional structure of the catalytic core of which resembles a hammerhead. Hammerhead ribozymes can hybridize with the target RNA through two recognition arms that are complementary to the target RNA, and cleave the target RNA in a sequence-specific manner.

HAIRPIN RIBOZYME

A ribozyme with a double helical region, a two-dimensional structure shaped like a hairpin, which is formed by base pairing adjacent to the sequences that are complementary with the target RNA. Hairpin ribozymes can hybridize and cleave the target RNA in a sequence-specific manner.

VIRUSOID

A small circular single-stranded RNA (∼350 nucleotides) that lacks a protein coat, and that is present in a protein coat of another helper plant virus. Its replication depends on the helper virus.

SMALL NUCLEAR RNA

(snRNA). A small RNA molecule that functions in the nucleus by guiding the assembly of macromolecular complexes on the target RNA to allow site-specific modifications or processing reactions to occur.

SMALL INTERFERING RNA

(siRNA). A short RNA (∼22 nucleotides) that is processed from longer dsRNA during RNAi. These short RNAs hybridize with mRNA targets, and confer target specificity to the silencing complexes in which they reside.

POST-TRANSCRIPTIONAL GENE SILENCING

(PTGS). A sequence-specific gene silencing phenomenon that might function as a defence mechanism against pathogenic nucleic acids such as viruses or transposons. RNAi is a form of PTGS in which dsRNA induces the degradation of homologous endogenous transcripts.

CHEMOTAXIS

A type of migration that is stimulated by a gradient of a chemical stimulant or chemoattractant.

MICRORNA

(miRNA). A non-coding RNA of 21–24 nucleotides, which is processed from an endogenous ∼70-nucleotide hairpin RNA precursor by the RNase-III-type Dicer enzyme. miRNAs are evolutionarily conserved molecules and are thought to have important functions in various biological mechanisms.

FLUORESCENCE RESONANCE ENERGY TRANSFER

(FRET). Process of energy transfer between two fluorophores. Can be used to determine the distance between two attachment positions within a macromolecule or between two molecules.

MOLECULAR BEACON

A nucleic acid probe with a stem–loop structure that has two different fluorescent dyes (a fluorophore and a quencher) at each end. Hybridization of the probe with the target unwinds the stem–loop structure and allows emission of fluorescent signals by uncoupling of the fluorophore and the quencher.

RNA-INDUCED SILENCING COMPLEX

(RISC). A multi-component, ribonucleoprotein complex that cleaves specific mRNAs that are targeted for degradation by homologous dsRNAs during the process of RNA interference.

SHORT HAIRPIN RNA

(shRNA). A hairpin-shaped RNA containing sense and antisense sequences connected by a loop sequence. shRNA is processed into 21–23-nucleotide siRNAs by Dicer, and can suppress gene expression by triggering the RNAi pathway.

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Akashi, H., Matsumoto, S. & Taira, K. Gene discovery by ribozyme and siRNA libraries. Nat Rev Mol Cell Biol 6, 413–422 (2005). https://doi.org/10.1038/nrm1646

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