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unit (Rodriguez et al. 2004). Finally, some pri-miRNAs are found in intergenic
regions whereby they contain their own promoters and are independently tran-
scribed (Zeng 2006).
Transcription of most pri-miRNAs is RNA polymerase II-dependent (Lee et al.
2004). Notable exceptions include the MHV68 miRNAs, whose expression is
believed to be driven by a pol III promoter (Pfeffer et al. 2005). Pol II-dependent
pri-miRNAs possess 5′ cap structures and are polyadenylated (Cullen 2004). Due
to the enormity and diversity of pol II-associated transcription factors, pol II-dependent
transcription allows for the exquisite temporal and spatial control characteristic of
miRNAs.
Following transcription, an approximately 65-nt hairpin with a 2-nt 3′ overhang
is excised from the pri-miRNA by the nuclear Microprocessor (Gregory et al.
2004). Microprocessor consists of the RNase III type endonuclease Drosha in com-
plex with the cofactor DGCR8 (Han et al. 2004). These two components of
Microprocessor are both necessary and sufficient to affect cleavage in vitro. Using
mutagenesis and in vitro processing assays, it was determined that Microprocessor
recognition of pri-miRNA is through the ssRNA flanking strands and the structural
motif of the hairpin (Han et al. 2006). Recent studies suggest that pri-miRNA
processing to mature miRNAs is a regulated step in miRNA biogenesis. Mature
miRNAs have been found in fully differentiated cells, while the pri-miRNAs accu-
mulate in undifferentiated cells (Thomson et al. 2006). Additionally, in human
tumors pri-miRNAs are expressed at high levels while the formation of mature
miRNA is downregulated (Thomson et al. 2006). This suggests that there may be
additional cofactors modulating either substrate specificity and/or activity of
Microprocessor.
Following conversion of pri-miRNA to pre-miRNA, Exportin-5, in a Ran-GTP-
dependant manner, exports the pre-miRNA from the nucleus to the cytoplasm
(Lund et al. 2004). A cytoplasmic RNase III type endonuclease, Dicer, then
removes the stem loop from the pre-miRNA converting it to a 22-bp dsRNA with
2-nt 3′ overhangs on each strand (Carmell and Hannon 2004). Evidence suggests
that pre-miRNA processing to mature miRNAs is also subject to regulation. It is
unclear whether this is due to regulation of pre-miRNA export from the nucleus, or
due to the presence of cofactors, which may influence substrate specificity, and/or
activity of Dicer cleavage (Lund and Dahlberg 2006; Obernosterer et al. 2006).
Typically, one strand of the 22-bp dsRNA, designated the guide strand, is fated
to pair with a target mRNA to facilitate translation inhibition or cleavage of the tar-
get mRNA. The guide strand is chosen based on lower thermodynamic stability at
its 5′ end in the duplex RNA (Khvorova et al. 2003; Schwarz et al. 2003).
Incorporation of the guide strand in the miRISC enables identification of target
mRNA for either translation inhibition or cleavage.
Mammalian miRISC is at minimum comprised of Dicer, Argonaute proteins,
TRBP, and PACT (Rana 2007). In general, translation inhibition occurs if there is
imperfect complementarity of the guide strand with the target mRNA. Cleavage
occurs if the complementarity between the guide strand and the target mRNA is
perfect. Nucleotides 2-7 of the miRNA constitute the seed region of the miRNA
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