Introduction:

This paper is investigating the effects of miRNA disruption on protein expression and splicing
patterns. MiRNAs help regulate gene expression post-transcriptionally by targeting mRNAs for
degradation or preventing translation of mRNA.

Background:

miRNA: non-coding RNA molecule which functions as a post transcriptional regulation of gene
expression by base pairing with complementary mRNA
Drosha
Dicer

CELF family splicing regulators

Alternative Splicing is a regulated process during transcription that results in a single gene
coding for multiple proteins
Protein Isoforms - expressed temporally and spatially
CUGBP1
CUGBP2

Heart Development -

Mechanism of MiRNAs:

1) Transcription of palindromic DNA by RNAPII ---> miRNA forms long hairpin

2) Drosha cleaves the RNA into smaller, double stranded RNA hairpins
3) Dicer opens the hairpin, ‘activating’ the miRNA
4) Formation of the RISC complex with ss-RNA and Argonaute
5)MiRNA 23a/b in the myocardium downregulate translation of CUGBP and ETR-3 like factors
during heart development
6) (hypothesis) CUGBP1 and CUGBP2 are splicing regulators in charge of altering isoforms of
key regulatory genes in the myocardium during development of the hear: high levels favor the
embryonic splicing patterns of target proteins.

In this study, Step 3 was blocked so as to interrupt the formation of miRNAs and all subsequent
steps, by Tamoxifen-inducible Cre/lox-P knockout of Dicer. Addition of Tamoxifen causes
transcription and translation of the Cre protein, which binds to lox-p constructs surrounding the
Dicer gene and excises that gene so that Dicer cannot be expressed. The promoter used for
Cre, a-Myosin heavy chain, is specific to cardiomyocytes so that this effect is targeted to only
one tissue and does not disrupt the entire organism.
MiRNA23a/b were recognized as candidates through expression patterns and homology with
the 3’ UTR of CELF mRNAs.

Figure 1:

a) Western blot verifying that Tam addition causes decrease in Dicer. Dicer levels do not drop to
zero because of possible non-cardiomyocytes and possibly a lack of complete excision.

B) northern blot showing pre-miRNA accumulation upon Dicer knockout. U6 snoRNA is a

loading control.

C) shows that after 12 wks, all Tam+ individuals died.

D) Increase in heart size upon Dicer knockdown

E) malformation of heart tissue

F) increase in CUGBP1/2 after Tam treatment

G) shows change in splicing pattern of one exon is specific to Dicer knockout; other cardiac
injuries don’t show the same effect.

Figure 2
A) Western blot showing Tam-induced upregulation of CUGBP1/2, PTB, Fox-1, Fox-2 isoform 2,
Tial, hnRNP (What does this control again?), all of which are splicing regulators. The others are
also splicing factors, showing that the effect is specific to certain factors.

B) mRNA levels of CUGBP1/2 are unaffected by Dicer, indicating post-transcriptional control of

the increase

C) Basal splicing regulators are unaffected, further confirmation that this is a targeted
phenomenon

D) Were these results really localized to the cardiomyocytes or due to the influx of inflammatory
cells?
Top panel: Fox-1 and C1, C2 localize to the nucleus that also stain w/Desmin, a marker
of cardiomyocytes. Absence of green in merge shows that they are only found in this cell type.

E) Yellow shows E14 (Embryonic day 14 levels) for Ank2, Sorbs1, H2afy, Mfn2, Mtmr3, all
exons regulated by CELF proteins

Black shows normal pattern at various stages. The dicer KO mice showed significant
differences from the normal and reverted closer to the embryonic splicing patterns.

F) This effect is missing in exons not subject to CELF regulation (targeted)

Figure 3:

A) shows the 3 miRNA binding sites on CUGBP1 and 2 on CUGBP2 mRNA. These were sites
used and/or mutated in the following figure.

B) upregulation of miRNA - 23 a/b corresponds with decrease in CELF protein levels, showing
the possibility of a causal relationship.

C Quantification of B

D) A luciferase reporter was attached to each 3’ site, a normal and mutant (green) version.
Mirna 23 b was enriched in the red and green columns. NS is a non-binding miRNA (non-
silencing). Only the wild sites showed a decrease in luciferase, showing that miR-23b can
directly control translation of each of the putative sites of the 3’ UTR.

E Verification of miR-23b enrichment

F) Decrease in CELF but not control
G) again, no mRNA change (post-transcriptional control of CELF levels)

H) change in splicing pattern of CELF -regulated proteins

Figure 4

Rather than KO dicer, knock down miRNA 23a/b with an antagomir

A) verification via qPCR

B) mRNA of CUGBP1/2 is unchanged
C) increase in protein levels (western blots)

D) Quantification
E) change in splicing patterns after kd of mir23 a/b