Making siRNA with RNase III

Make siRNA with RNase III

Introduction | Bacterial RNase III | Dicer

Ribonuclease III (RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD).

Endoribonuclease III (RNase III) was the first double-stranded RNA (dsRNA) specific endoribonuclease to be discovered in E. coli and although it is not required for viability of bacterial cells (B. subtilis seems to be an exception) it is conserved even in some very small genomes, as for instance in Mycoplasma genitalium.

RNase III catalyzes the cleavage of ribosomal RNA (rRNA) precursors during maturation of rRNA (1). For a short review on RNase III and its various substrates see also (15). In Rhodobacter capsulatus, 23S rRNA is fragmented: 14 and 16S fragments form the functional equivalent of intact 23S rRNA. An extra stem-loop is removed from 23S rRNA by RNase III (9, 13). Fragmentation at other positions within 23S rRNA also occurs (13). Furthermore, a significant number of mRNAs, including the message for RNase III (rnc), are cleaved by the enzyme in E. coli. Additional functions include processing of snRNA and snoRNA (in eukaryotes) (2; 3). Recently, human RNase III has also been studied more intensively (10). In eukaryotes RNase III seems to be essential.

Cleavage site determination by bacterial RNase III seems to be carried out via so called "antideterminants" adjacent to the cleavage site (6). This mechanism of cleavage site selection is not universally true for all RNase III substrates and enzymes (11, 12, 13). A consensus sequence for cleavage specificity is only poorly defined. A substrate of at approximately 20 bp of dsRNA (two turns of an A-form helix) is needed for cleavage. The enzyme needs divalent cations for cleavage (1; 5; 7). The E. coli enzyme is a homodimer of about 50 kDa (ca. 25 kDa for each subunit) and comprises only 0.01% of total cell protein; it possesses a C-terminal dsRNA binding domain (dsRBD) of about 70 amino acids (4) and an N-terminal catalytic domain.

RNase III is coded by the rnc gene and the expression shows autoregulation due to a stem loop structure upstream of rnc. In R. capsulatus the rnc gene overlaps the preceding lep gene (leader peptidase) by 2 nt and both form an operon with the promotor of rnc lying upstream of lep (8). RNase III orthologs in eukaryotes are Rnt1p of S. cerevisiae and Pac1 of S. pombe (2; 3). The enzyme from S. cerevisiae has a different mode of cleavage site determination than the bacterial RNase III: it recognizes a conserved RNA-tetraloop (11, 12). RNase III enzymes do also have important functions in RNA interference (RNAi) in eukaryotes (15).

References:

Nicholson AW (1999) Function, mechanism and regulation of bacterial ribonucleases. FEMS Microbiol Rev 23 (3):371-90.
Kufel J, Dichtl B, Tollervey D (1999) Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS. RNA 5 (7):909-17.
Zhou D, Frendewey D, Lobo Ruppert SM (1999) Pac1p, an RNase III homolog, is required for formation of the 3' end of U2 snRNA in Schizosaccharomyces pombe. RNA 5(8):1083-98.
Kharrat, A., Macias, J., Gibson, T. J., Nilges, M. and Pastore, A. (1995) Structure of the dsRNA binding domain of E. coli RNase III; The EMBO Journal 14, pp. 3572-3584.
Li, Hong-Lin, Chelladurai, B. S., Zhang, K. and Nicholson, A. W. (1993) Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects; Nucleic Acids Res. 21, pp. 1919-1925.
Zhang K, Nicholson AW (1997) Regulation of ribonuclease III processing by double-helical sequence antideterminants. Proc Natl Acad Sci U S A 94(25):13437-41.
Nicholson, Allen W.(1996) Structure, Reactivity and Biology of Double-Stranded RNA; Progress in Nucleic Acids Research and Molecular Biology 52, pp. 1-65.
Rauhut, R., Jäger, A., Conrad, C. and Klug, G. (1996) Identification and analysis of the rnc gene for RNase III in Rhodobacter capsulatus. Nucleic Acids Research, 24, pp. 1246-1251.
Conrad C, Rauhut R, Klug G (1998) Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli. Nucleic Acids Res 26(19):4446-53.
Wu H., Xu H., Miraglia L. J., Crooke S. T. (2000) Human RNase III is a 160 kDa protein involved in preribosomal RNA processing. J. Biol Chem 275:36957-65.
Chanfreau G., Buckle M., Jacquier A. (2000) Recognition of a conserved class of RNA tetraloops by Saccharomyces cerevisiae RNase III. Proc Natl Acad Sci USA 97:3142-3147.
Nagel R., Ares M. Jr. (2000) Substrate recognition by a eukaryotic RNase III: The double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5´-AGNN-3´tetraloop. RNA 6:1142-1156.
Evguenieva-Hackenberg E., Klug, G. (2000) RNase III Processing of intervening sequences found in helix 9 of 23S rRNA in the alpha subclass of proteobacteria. J Bacteriol 182: 4719-4729.
Conrad, C., Evguenieva-Hackenberg, E., Klug, G. (2001) Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III. FEBS letters 509/1: 53-58.

Bacterial RNase III

Dicer

Dicer, an enzyme can produce putative guide RNAs was first discovered in 2001. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

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