Biogenesis of Mammalian miRNA - MicroRNAs as Tools in Biopharmaceutical Production - page 19

Similar to Drosha in the nucleus, Dicer interacts with other proteins forming larger
complexes involved in loading of the RNA induced silencing complex (RISC). The
dsRNA binding protein TAR RNA binding protein (TRBP) and protein activator of
PKR (PACT) are not required for pre-miRNA processing by Dicer, but contribute
instead to the stability of the complex and formation of the RISC (Haase et al. 2005 ;
Lee et al. 2006 ; Chendrimada et al. 2005 ; Melo et al. 2009 ; Paroo et al. 2009 ). The
mature miRNA duplex is therefore determined both by recognition of the pri-miRNA
by Drosha in the nucleus and cleavage by Dicer in the cytosol. The mature miRNA
may be located on either side of the pre-miRNA hairpin duplex.

Targeting is Influenced by The 5 End of The MicroRNA

2.5

The selection of cleavage sites by Drosha and Dicer may produce miRNA with
multiple isoforms depending on the sequence of the 5 and 3 ends (Carthew and
Sontheimer 2009 ). As explained below, differences in nucleotide composition can
alter the “seed” sequence ultimately affecting which protein-coding mRNAs are tar-
geted by the mature miRNA (Chiang et al. 2010 ). The ability of miRNA to repress
translation is largely a function of the thermodynamic stability of base-pairing be-
tween the miRNA and complementary sequences generally found in the 3 UTR of
target mRNA. In particular, nucleotides at positions 2-8 at the 5 end of the miRNA
comprise the so-called “seed” sequence for miRNA target recognition (Doench and
Sharp 2004 ). Although both the 5 and 3 ends of miRNA can vary depending on
the addition or deletion of 1-2 nt, alterations in the 5 sequence can alter the seed
sequence resulting in potential changes in miRNA target specificity. Additionally,
thermodynamic stability of the 5 and 3 ends may affect which strand of the duplex
is incorporated into the RISC (Krol et al. 2010 ). Differential selection of cleavage
sites by Drosha and Dicer can therefore alter both the stability of the duplex and
determine which strand is incorporated into the RISC as a mature miRNA.

2.6 RISC Assembly

Following cleavage by Dicer, the 22-nt RNA duplex containing both the mature

miRNA (guide strand) and the passenger strand (also referred to as the miRNA ∗
strand) is incorporated into the RISC. In order to function in miRNA-mediated reg-
ulation of gene expression, the duplex is separated and the guide strand is retained
in the RISC whereas the passenger strand is removed. RISC assembly occurs in two
steps: first,

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22-nt RNA duplexes are incorporated into Argonaute (Ago) proteins
and second, RNA duplexes are unwound or separated within Ago proteins (Kawa-
mata and Tomari 2010 ). Argonaute proteins may be divided into three groups based
on sequence similarity and the nature of their substrates: the AGO subfamily, the
Piwi subfamily, and the C. elegans -specific WAGO subfamily (Czech and Hannon

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