RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 2

Fire and Mellow showed that the introduction of exogenous long double-stranded
RNA (dsRNA) into the nematode worm Caenorhabditis elegans triggers potent
silencing of genes that share perfect sequence complementary to the introduced
dsRNA, a silencing effect far superior to what was typically seen using single-
stranded antisense RNA, the prevalent gene silencing tool at the time.
Several important observations quickly followed; the exogenous long dsRNA
was found to be enzymatically processed into shorter 20-25-base pair (bp) dsRNAs
acknowledged as the true effectuators of RNAi [ 2- 4 ] . The subsequent observation
that exogenous, synthetic 21-bp dsRNAs could trigger highly effective and sequence-
specific gene silencing upon introduction in the cytoplasm in almost all investigated
eukaryotic organisms provided researches with an extremely potent gene silencing
tool [ 5 ]. These small dsRNA species, coined small interfering RNAs (siRNAs),
marked the birth of a completely new silencing mechanism and the entry into the
RNAi era of molecular biology and medicine. Besides being established as an ubiq-
uitous tool in basic research, the number of RNAi-based preclinical and clinical tri-
als has increased rapidly over recent years providing optimism for successful clinical
translation [ 6, 7 ], a high achievement for such a fledgling technology.

1.2 RNAi Pathways

1.2.1 The Natural Biological Functions of RNAi Interference

As presented throughout this chapter, the unprecedented power of RNAi as a gene
silencing tool originates from harnessing natural gene silencing pathways to effec-
tuate efficient degradation of any chosen target mRNA. All key enzymes in the
RNAi pathway are broadly conserved throughout the eukaryotic clade, suggesting
the existence of a minimized version of the RNAi machinery in the last common
ancestor of eukaryotes [ 8 ]. Here, RNAi is hypothesized to have served primarily as
an ancient defense mechanism that functions in cis to endonucleolytically cleave
and degrade exogenous long cytoplasmic dsRNA originating from viral infection
[ 9- 11 ] , aberrantly expressed transgenes [ 10, 12- 14 ] , mobile genetic elements [ 15,
16 ], or aberrant processing of endogenous mRNA [ 17 ]. In higher eukaryotes, how-
ever, the RNAi machinery has evolved to be a key aspect in the general regulation
of endogenous gene expression. Here, a class of endogenous 20-25 nucleotide sin-
gle-stranded RNAs, known as microRNAs (miRNAs), are found to regulate the
stability and translation of mRNAs in trans upon directly base pairing to their
3¢untranslated regions (3¢UTRs). The first example of a miRNA, a noncoding gene
termed lin-4 encoding a small RNA species of ~22 nt, was identified in the nema-
tode worm C. elegans in 1993 [ 18 ]. It was hypothesized that lin-4 represses transla-
tion of the lin-14 gene by base pairing partially with putative target sites positioned
in the 3¢UTR of the lin-14 mRNA. It took almost 7 years before the second miRNA,
let-7 , was shown to regulate the lin-41 mRNA through a similar mechanism.
Interestingly, let-7 was found to be evolutionary conserved in a wide range of animal
species indicating a more general role of miRNA in gene regulation [ 19- 21 ] .