Biology Reference
In-Depth Information
A miRNA expression reporters
i. Enhancer traps
ii. Direct enhancer : reporter transgenes
P
element with
lacZ
,
Gal4, etc.
Reporter :
lacZ
,
GFP
,
DsRed
,
Gal4
etc.
Terminator
Endogenous CRMs
Cloned CRM
Report transcriptional output of endogenous miRNA locus
Report activity of one or more miRNA enhancers
B miRNA activity reporters
i. miRNA “sensor” transgenes
ii. 3'UTR reporters
Constitutive
promoter
(tubulin)
Constitutive
promoter
(tubulin)
Reporter
(
GFP
)
Reporter
(
GFP
)
Terminator
Terminator
Perfect miRNA target sites
miRNA target sites
Multimer of perfect complementary sites to a miRNA
Reports on miRNA function
3'UTR of miRNA-regulated gene
May be regulated by several miRNAs
C Use of miR sensors / reporters
i. Ectopic miRNA test
ii. Loss of miRNA test
miRNA
Reporter
Clone
Sensor
-
/
-
+/+
-
/
-
+/+
+/
-
+/
-
Repression of reporter (green)
in miRNA-expressing domain (red)
Induction of homozygous miRNA mutant (
-
/
-
) and
wildtype (+/+) clones in heterozygous animal (+/
-
)
Derepression of miRNA sensor mutant cells
Figure 8.4
Reporters of miRNA expression and activity. miRNAs are transcribed as
long primary transcripts, whose mature
22nt products guide Argonaute proteins
complexes to target transcripts. Note that the transcription of a miRNA locus does
not necessarily report directly on cells in which the miRNA is active. (A) miRNA
expression reporters are the same as those used to report expression of protein-coding
genes. (i) Putative regulatory sequences are identified by enhancer traps (insertion
of transposon with a marker gene, often lacZ or Gal4) where the endogenous cis-
regulatory modules (CRMs) drive expression of the marker. (ii) Cloned CRMs can
also be used to analyze miRNA gene expression; however, these may only report on a
subset of the full transcriptional control of a miRNA. (B) Sensors of miRNA activity
typically use a marker gene (often GFP) bearing sequences complimentary to the
miRNA of interest (i) or with the 3
0
UTR of a gene of interest (ii) cloned downstream.
Sensor activity is inverse to that of the miRNA: low sensor expression equates to high
