Biology Reference
In-Depth Information
2. The Discovery of miRNAs
2.1. lin-4 miRNA
Development of
C. elegans
progresses through four larval stages before
reaching adulthood. The temporal fate of each cell division has been
mapped and genes that regulate the timing of these events are part of the
heterochronic pathway (
Sulston and Horvitz, 1977
). Mutations in hetero-
chronic genes cause either precocious development, where later larval cell
fates occur prematurely, or retarded development, where early larval cell
fates reoccur in later stages (
Ambros, 1989; Ambros and Horvitz, 1984;
Chalfie
et al.
, 1981
). These types of mutations cause various developmental
abnormalities, some of which result in lethality. While investigating the
genes important for larval development, researchers found that
lin-4
loss-of-
function (lf ) mutations recapitulated phenotypes observed in worms with
lin-14
gain-of-function (gf ) mutations (
Ambros, 1989
). These mutant
worms repeat L1 cell fates, which cause the absence of adult structures
like the vulva and differentiated hypodermal seam cells (
Chalfie
et al.
,
1981
). Loss of
lin-14
activity has the opposite phenotype, where later larval
fates are precociously expressed in the first larval stage (
Ambros and Horvitz,
1984
). Additional genetic and molecular experiments led to the model that
LIN-14 protein promotes the first larval stage cell fates and then is down-
regulated by
lin-4
activity so that later larval fates can proceed (
Arasu
et al.
,
1991; Ruvkun and Giusto, 1989
).
The surprising identity of the
lin-4
gene product revealed a novel
mechanism for regulation of
lin-14
. Through arduous mapping and genetic
rescue experiments, researchers in the Ambros lab narrowed down the
location of
lin-4
to a 693nt region, which was later shown to contain the
primary lin-4 transcripts, that lacked apparent protein-coding potential
(
Bracht
et al.
, 2010; Lee
et al.
, 1993
). Instead, this region was found to
express 22 and 61nt RNA products that are now recognized as the mature
and precursor forms of lin-4 miRNA, respectively. Since the Ruvkun lab
had established that
lin-14
is regulated at the posttranscriptional level
through elements in its 3
0
UTR, the possibility of lin-4 base-pairing to
these regions was realized by both labs (
Lee
et al.
, 1993; Wightman
et al.
,
1993
). There are seven potential lin-4 target sites in the
lin-14
3
0
UTR that
are predicted to form nonidentical partial duplexes (
Wightman
et al.
, 1993
).
Using genetic mutants, molecular experiments, and reporter assays, the
Ambros and Ruvkun labs defined a novel form of gene regulation whereby
the lin-4 RNA base pairs to specific sites in the 3
0
UTR of the
lin-14
mRNA, resulting in downregulation of LIN-14 protein expression (
Lee
et al.
, 1993; Wightman
et al.
, 1993
). Whether or not this mechanism of gene
regulation would be an isolated example was an open question since the
