Biology Reference
In-Depth Information
small RNA, lin-4, and its target gene,
lin-14
, were not obviously conserved
in species beyond
Caenorhabditis
.
2.2. let-7 miRNA
Four years after the discovery of lin-4 and its target, another protein-coding
gene in the heterochronic pathway was found to be under the direct control
of
lin-4
. The
lin-28
3
0
UTR contains a single
lin-4
binding site that mediates
repression of protein expression at the posttranscriptional level (
Moss
et al.
,
1997
). With two different genes regulated in a similar manner by the same
RNA, researchers suspected that other small RNA regulators might exist in
C. elegans
to control developmental timing
.
Characterization of mutations
that cause delayed temporal fates in the last larval stages led to the
let-7
gene
(
Fig. 1.3
A;
Reinhart
et al.
, 2000
). The
let-7
gene was originally named based
on the lethal phenotype where worms rupture through the vulva and die
before reaching adulthood (
Fig. 1.3
B;
Meneely and Herman, 1979
). Genetic
mapping, rescue experiments, and RNA expression analyses established that
let-7
was the second example of a tiny RNA gene in the developmental
timing pathway (
Reinhart
et al.
, 2000
). Like
lin-4
, the
let-7
gene expressed
an
21nt RNA that could potentially derive from a processed intramolec-
ular hairpin structure (
Lee
et al.
, 1993; Reinhart
et al.
, 2000
). Coinciden-
tally, studies of the
lin-41
gene in the same lab showed that mutations in this
heterochronic gene resulted in precocious adoption of differentiated cells
fates, a phenotype opposite to that of
let-7
mutants (
Fig. 1.3
A;
Reinhart
et al.
, 2000; Slack
et al.
, 2000
). Using a variety of biochemical and genetic
techniques, the let-7 RNA was demonstrated to regulate expression of
lin-
41
through two sites of partial complementarity in its 3
0
UTR (
Fig. 1.4
;
Reinhart
et al.
, 2000; Slack
et al.
, 2000; Vella
et al.
, 2004a
). Thus, the
accumulation of mature let-7 RNA at the end of the third larval stage
causes downregulation of LIN-41 protein expression, allowing the adop-
tion of later larval and adult fates.
2.3. let-7 miRNA in other organisms
Unlike
lin-14
, homologs of
lin-41
were found to exist in Drosophila,
mouse, and other animals, raising the question of whether its small RNA
regulator would also be conserved (
Slack
et al.
, 2000
). With whole-genome
databases becoming available, the Ruvkun lab identified sequences in the
Drosophila and human genomes that matched the mature let-7 sequence
(
Pasquinelli
et al.
, 2000
). Northern blot analyses of a wide range of animal
species showed that let-7 RNAs are present broadly in bilaterians, but
apparently not in basal metazoans or nonanimal organisms. Remarkably,
potential let-7 binding sites exist in the 3
0
UTRs of
lin-41
homologs in
several species and, in some cases, regulation by let-7 has been demonstrated
