Ribonuclease activity and RNA binding of recombinant human Dicer

Abstract

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated~21±23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg 2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer´dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.

Principles of micro-RNA production and maturation

“…Dicer is another RNaseIII-type enzyme, and it cleaves pre-miRNAs in the cytoplasm (Billy et al, 2001;Grishok et al, 2001;Hutva´gner et al, 2001;Ketting et al, 2001;Provost et al, 2002;Zhang et al, 2002). Most Dicer proteins possesses an approximately 130-amino-acid-long PAZ domain (Figure 3), which preferentially binds to single-stranded 3 0 ends of doublestranded RNAs (Lingel et al, 2003;Song et al, 2003;Yan et al, 2003;Ma et al, 2004).…”

The microRNA world: small is mighty

“…In that study, purified human Dicer was incubated with in vitro transcribed pre-let-7 RNA, and the products of the reaction were analyzed by northern blotting with probes recognizing let-7 and its complement, corresponding to the opposite strand of the pre-let-7 stem. Only a single , 22nt RNA was detected corresponding to let-7, implying that Dicer recognizes the authentic miRNA and cleaves the precursor only in one strand [36]. These results, however, should be interpreted with caution because recombinant Dicer was not sufficiently pure, representing only 25% of the total protein [36].…”

“…This latter possibility, however, is less likely because in vitro processing of pre-let-7 in Drosophila embryo extracts produces let-7 only [31]. It was also reported recently that recombinant human Dicer protein prepared from baculovirus-transduced insect cells is able to cleave synthetic pre-let-7 in vitro [36]. In that study, purified human Dicer was incubated with in vitro transcribed pre-let-7 RNA, and the products of the reaction were analyzed by northern blotting with probes recognizing let-7 and its complement, corresponding to the opposite strand of the pre-let-7 stem.…”

“…Only a single , 22nt RNA was detected corresponding to let-7, implying that Dicer recognizes the authentic miRNA and cleaves the precursor only in one strand [36]. These results, however, should be interpreted with caution because recombinant Dicer was not sufficiently pure, representing only 25% of the total protein [36]. It is thus possible that other factors required for proper pre-miRNA processing, and normally present in insect cells which contain the miRNA/RNAi machinery, copurified with recombinant human Dicer.…”

Emergence of a Complex Relationship between HIV-1 and the microRNA Pathway

“…The pre-miRNAs are subsequently exported to the cytoplasm via Exportin-5 (15)(16)(17)(18) and the base of their stem recognized by the PAZ domain of Dicer (19). Acting as an intramolecular dimer, the RNase IIIa and IIIb domains cleave the stem at the base of the loop to generate a miRNA:miRNA* duplexes (19)(20)(21)(22). Dicer was recently shown to operate with the transactivating response RNA-binding protein (TRBP) (23) within a pre-miRNA processing complex (24,25).…”