Biochemical characterization of human Dicer and its interacting partner TRBP

Over the recent years, RNA interference (RNAi) has emerged as a powerful
method to study the role of individual genes. However, the mechanism
underlying the gene silencing by the double-stranded RNA (dsRNA) is still not
fully understood. RNAi is initiated when dsRNA is processed by RNase III
endonuclease Dicer into short interfering RNAs (siRNAs), of 21 to 22 nucleotides
in length. SiRNAs are then incorporated into RNA-induced Silencing Complex
(RISC) that by base-pairing targets messenger RNA for degradation. Dicer is
also involved in processing of precursors of the small regulatory RNA species,
microRNAs (miRNAs). MiRNAs are encoded in the genome and are implicated in
gene expression regulation in various cellular processes. After maturation by
Dicer, miRNAs are incorporated into RISC-like complexes that in animals
imperfectly base-pair with the target mRNA and lead to inhibition of translation.
This thesis focuses on Dicer, the central protein involved in both RNAi and
miRNA pathways.
Detailed study of the ribonuclease activity of human Dicer and its
ancestral prototype, bacterial RNase III, are described in the first experimental
chapter of this thesis. The common model for dsRNA cleavage by the RNase IIIclass
enzymes is proposed. The use of mutagenesis to investigate the catalysis
revealed that Dicer and bacterial RNase III contain a single compound catalytic
center. Both RNase III domains of Dicer contribute to the dsRNA cleavage
reaction. The results obtained in this study have proved the then-accepted model
of RNase III catalysis to be inadequate. We demonstrated that instead of the two
catalytic centers as proposed in the old model, both E. coli RNase III and Dicer
contain one compound catalytic center that generates products with 2-nt 3’
overhangs. In silico modeling of the dsRNA substrate into 3D crystal structure
coordinates of the bacterial RNase III offered additional support to our
interpretation. Together with other data, a new model was proposed according to
which Dicer functions as an intramolecular pseudodimer of its two RNase III
domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.
Second chapter describes dsRNA binding domain (dsRBD)-containing
protein, TRBP, that was found to associate with Dicer in mammalian cells and in
vitro. We show that TRBP is required for optimal RNA silencing mediated by
siRNAs and endogenous miRNAs, and that it is involved in efficient processing of
pre-miRNAs. Since TRBP had previously been described as the inhibitor of the
interferon-induced double-stranded RNA-regulated protein kinase PKR, the
TRBP-Dicer interaction raises a possibility of the connection between RNAi and
interferon-PKR pathways.
DsRNA binding properties of human Dicer dsRBD are described in the
third chapter. We have found that this domain has the propensity to bind dsRNAs
of different lengths. Surprisingly, it displays hardly detectable affinity for siRNAs.
This observation suggests that the dsRBD might be involved in substrate binding
during Dicer cleavage reaction and take part in substrate/product discrimination
preventing the enzyme from sequestering its own product.
The two supplementary chapters contain the work performed in
collaboration with other laboratories. We show that like its Drosophila counterpart,
human Dicer is able to form complexes with siRNAs both in vitro and in vivo.
These results indicate that also in mammals Dicer could function downstream of
the dsRNA cleavage step and could take part in RISC assembly. The other
supplementary chapter describes RNAi connection with chromatoid bodies
during spermatogenesis. We show that Dicer and components of the RISC-like
complex (Ago and miRNA) are concentrated in chromatoid body. We also
demonstrate that Dicer directly interacts with the RNA helicase MVH (mouse
Vasa homolog) that is the germ-line specific chromatoid body component. Our
findings suggest that the chromatoid body might function as a subcellular
concentration site for the miRNA pathway components during spermatogenesis.