Biology Reference
In-Depth Information
regions are involved, mutations do not alter or destabilize the
protein itself, but most likely interfere with posttranscriptional
processes. Finally, demonstration that the mysterious “RNA-RNA
interaction” equals complementary binding of miRNA lin-4 to lin-
14 mRNA suggested a whole new noncoding RNA-world to be
investigated [
6
,
7
]. The second miRNA discovered as recently as in
the year 2000 is let-7 encoding a 21 nt small RNA also comple-
mentary to elements in the 3
-UTR of lin-14 among others, like
lin-28 and lin-41 [
8
]. In
C
.
elegans
let-7 functions as a heterochro-
mic switch gene between larval and adult stage coordinating
developmental timing. Surprisingly, both let-7 and lin-41 are
evolutionarily conserved with homologues readily detected in
mollusks, fl ies, mice, and humans [
9
]. Database searches (BLASTN)
for matches to let-7 RNA reveal DNA segments from
Drosophila
melanogaster
as well as from the human genome sequence. As for
precursor transcripts of let-7 RNA, similar stem-loop second-
ary structures are predicted for
Caenorhabditae
,
Drosophila
, and
H
.
sapiens
. Interestingly, only this similar RNA fragment can be
detected in most species, emphasizing that let-7 may be effi ciently
processed from a putative precursor to a mature form of miRNA.
Raising proof indicates that let-7 is essential for gene regulation in
late temporal transitions across animal phylogeny.
Known by now, mature miRNA mostly derives from one arm
of larger precursor miRNA which forms imperfect stem-loop
structure [
10
]. Biogenesis of miRNA starts within the nucleus:
miRNAs are encoded in the genome as pri-miRNA [
11
]. These
long primary transcripts extend both 5
′
′
and 3
′
from the miRNA
sequence and contain a cap structure at the 5
′
-end and a polyade-
nylated 3
-end. Primary transcript is successively processed by two
ribonucleases (RNase III types) to its mature form [
12
]. Two
sequential reactions trim the transcript into mature miRNA.
Processing basically depends on the formation of miRNA into a
stem-loop structure [
10
]. First, pri-miRNAs are processed into ~70 nt
precursors (pre-miRNA) by cellular multiprotein complex pre-
dominantly located in the nucleus. Its core components are RNase
III enzyme Drosha and double-stranded RNA-binding domain
DGCR8/Pasha. Drosha cleaves cotranscriptionally generating
pre-miRNA with an RNase III characteristic 2 nt 3
′
-overhang.
Effi ciency of Drosha processing depends on pre-miRNA's terminal
loop size, stem structure, and fl anking sequence of the Drosha
cleavage site [
13
]. The following export from the nucleus into the
cytoplasm by exportin 5 (Exp5) is initiated by 3
′
-overhang recog-
nition. Inside the cytoplasm, the hairpin pre-miRNA is cleaved by
second RNase III enzyme Dicer into a small, imperfect dsRNA
duplex. Double-stranded RNA now consists of both mature
miRNA strand and its complementary strand ranging from 21 to
25 nucleotides. Dicer enzyme is characterized by several domains
in specifi c order. From the amino-to-carboxy terminus it contains
′
