Biomedical Engineering Reference
In-Depth Information
and cell line development strategies. However, whilst the potential to manipulate
microRNAs for biotechnological benefit holds much promise, it has a number of
pitfalls that are partly due to the fundamental principles of miRNA:mRNA target
recognition and the difficulty in predicting to what degree the titration/manipulation
of a particular microRNA will impinge on its one or many mRNA targets. In this
chapter, we provide a brief account of the in silico (e.g. target prediction software)
and experimental (e.g. reporter genes and sponge vectors) tools available for re-
searchers working in microRNAs and the manipulation of mammalian cell factories.
In considering the wide influence of microRNAs, we place a special emphasis on
the cellular effects associated with the manipulation of microRNA and high yield of
protein production with reference to use in Chinese hamster ovary (CHO) expression
systems.
1.2
Biogenesis and Evolution
MicroRNAs were first described in C. elegans as an antisense RNA regulating the
level of LIN-14 protein (Lee et al. 1993 ). The number of small RNAs described has
since exploded. As an example the online repository miRBase (http://mirbase.org/)
release 18.0, November 2011 contains 18,226 entries representing hairpin precursor
miRNAs, expressing 21,643 mature miRNA products in 168 species. These include
1,527 in human, 741 in mouse, 408 in rat and surprisingly only two have been
deposited for the hamster.
The maturation pathways of microRNAs are now well defined and the field is
extensively reviewed elsewhere (Kim et al. 2009 ; Lau and MacRae 2009 ; Westholm
and Lai 2011 ; Winter et al. 2009 ). In summary, microRNA biogenesis is initiated by
the transcription (usually by Pol II) of hairpin-containing RNAs, which are known
as pri-miRNAs or mIRtrons in the case of intronic miRNAs. In the first scenario, the
primary transcripts are cleaved into 55-70 nt hairpins, called pre-miRNAs, by the
nuclear RNase III multicomplex Drosha and its dsRBD partner DGCR8 (Han et al.
2004 ; Lee et al. 2003 ) and is subsequently exported to the cytoplasm. For intron-
derived microRNAs, the consensus is that Drosha is substituted by splicing although
cleavage of the miRNA hairpin can occur prior to the splicing of mRNA (Kim
and Kim 2007 ). Subsequently, miRtrons, which reside in the cytoplasm, undergo a
process known as debranching before adopting a pre-miRNA-like structure (Ruby
et al. 2007 ). Both pre-miRs are then further shortened by Dicer RNase III to yield
a 22 nt miRNA known as the miRNA/miRNA* duplex, which exhibit characteristic
3 overhangs at either end (Hutvagner et al. 2001 ; Kim et al. 2009 ). The strand that
accumulates to a higher level is referred to as the “mature” miRNA, while its less
abundant partner is referred to as the miRNA* or “star” strand. The abundant strand
is thought to be preferentially incorporated into an Argonaute (AGO) protein, which
acts as the core of an effector complex that is routed by the small RNA to targets and
the remaining strand is degraded.
The enzymes involved in the biogenesis of microRNAs are broadly conserved and
many of the bilaterian animal mature miRNAs are also phylogenetically conserved;
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