RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 3

Accordingly, thousands of miRNAs have since been identified, a task greatly
enhanced by next generation sequencing techniques (see http://www.mirbase.org ) ,
and the RNAi machinery has indeed proved widespread and important in control-
ling gene expression in nearly all aspects of cellular metabolism.

1.2.2 RNAi Pathways: Step by Step

Considering the essential roles for RNAi pathways in organism homeostasis, it is
clear that the development of safe RNAi-based therapeutics requires a thorough
understanding of the RNAi pathways by which eukaryotic organisms regulate their
transcriptome (Fig. 1.1 ). In essence, any artificial RNAi trigger employed, either
introduced into the cytoplasm as synthetic siRNAs or transcribed in the cell nucleus
from artificial RNAi vectors, will have to be carefully engineered to structurally
mimic endogenous RNA intermediates in the RNAi pathway in order not to inter-
fere with normal miRNA function. This section will, therefore, highlight key aspects
of the maturation and function of miRNAs in order to establish the ground rules
under which RNAi-harnessing approaches must comply.
Overall, the RNAi pathway has the potential to respond to a variety of RNA
substrates of both endogenous origin, that is, primary miRNA precursors (pri-
miRNAs) transcribed in the cell nucleus and exogenous origin such as cytoplasmic,
foreign dsRNAs, e.g., viral RNA, siRNAs, etc. The terms “miRNA pathway” and
“siRNA pathway” are often encountered in the literature to distinguish RNA sub-
strates of endogenous and exogenous origin, however, in essence all mature RNAi
triggers (miRNA and siRNA produced by the RNAi proteins from nuclear miRNA
precursor and cytoplasmic dsRNA, respectively) are functionally identical once
incorporated into the effector protein complex RISC in the cytoplasm [ 22, 23 ] .
Concurrently, multiple entry routes into the RNAi pathway can be exploited for
gene silencing therapeutics.
Focusing first on endogenous RNAi substrates, the majority of miRNAs are tran-
scribed as primary transcripts (pri-miRNA) in the nucleus by RNA polymerase II
and are situated either intronically in coding mRNA or as separate transcription
units [ 24, 25 ]. The pri-miRNA is hairpin structured and is initially recognized by
the bipartite complex, the microprocessor, composed of the endonucleolytic enzyme
Drosha and its cofactor DGCR8 [ 26, 27 ]. The microprocessor crops the hairpin
structure out of its flanking sequence, resulting in a 50-70-nt hairpin RNA, termed
the precursor miRNA (pre-miRNA). Drosha is composed of dual RNase III domains
responsible for the endonucleolytic cleavage, and the resulting product consists of a
5¢ phosphate and 3¢ hydroxyl group. Moreover, from a structural point of view, the
Drosha-processed pre-miRNA typically has two unpaired nucleotides in the 3¢ end,
referred to as a 2-nt 3¢ overhang. Basically, microprocessor products (with a few
exceptions) serve as bona fi de substrates in the downstream biogenesis pathway;
therefore, the specific pri-miRNA recognition by the microprocessor is a defining
step in miRNA biogenesis although the exact determinants of microprocessor rec-
ognition are still debated.