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What are minigenes?

A minigene contains a genomic fragment including the alternative exon(s) and the surrounding introns as well as the flanking constitutively spliced cloned in a eukaryotic expression vector.
Thus, the transfected minigenes should contain all RNA-elements necessary to show the same alternative splicing pattern as the corresponding endogenous alternatively spliced gene when compared in a specific cellular environment.

Oliver Stoss, Peter Stoilov, Annette M. Hartmann, Oliver Nayler, Stefan Stamm; Brain Research Protocols, 1999

 
         
 
The in vivo minigene approach

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type on alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections.

Drugs that change alternative splicing

The use of RNA-binding molecules as antibiotics, such as gentamicin, chloramphenicol, and tetracycline illustrates that drugs can be targeted against RNA and/or RNA binding proteins. High-throughput screens and testing of substances in model systems identified more substances that change splice site selection. The substances fall into several categories, including HDAC inhibitors, kinase and phosphatase inhibitors, as well as cAMP antagonist and agonists. The currently known substances are reviewed in (Sumanasekera et al., 2008 (Biochem Soc Trans. 2008)) and listed here.

The Alternative Splicing Database

The Alternative Splicing Database (ASD) Project aims to understand the mechanism of alternative splicing on a genome-wide scale by creating a database of alternative splice events and the resultant isoform splice patterns of genes from human, and other model species.

Visit the Database http://www.ebi.ac.uk/asd/

 
         
Figure legend

Summary of minigenes that have been used to analyze alternative splicing patterns in vivo. The structures of the various genes are schematically indicated, however, the drawings are not to scale. The various genes are sorted according to their splicing mechanism (A-H). Exonic splicing enhancers/silencers (ESE/ESS) or intronic splicing enhancers/silencers (ISE/ISS) are indicated by triangles marked with a "+" or with a "-", respectively. In some cases, special abbreviations were used for well characterized cis-elements:

DCS: downstream control sequence

ICR: intronic control region

MSE: muscle specific splicing enhancer

IRE: intron regulatory element

PTB: Polypyrimidine tract binding protein binding site.

The tip of the triangle points towards the resulting splicing pattern.

cis-elements containing identical sequence elements are marked by the same color. Identified tissue specific trans-acting factors are shown on top or bottom of the minigenes, depending on the stimulatory or inhibitory effect, respectively. When a direct correlation between binding of a splicing factor to a cis-element and a change of splicing patterns has been demonstrated, cis-elements and trans-acting factors are shown in the same color.

 
A: minigenes containing one cassette exon
# name species minigene reference
1 GABAAγ2
24nt Exon
rat

2 clathrin light chain B exon EN rat

3 src
exon N1
mouse

4 NCAM
exon 18
mouse

5 MHC-B
exon N30
human

6 agrin
exon Y
mouse

7 tau,
exon 6
human

8 fibronectin
EIIA
mouse
9 fibronectin
EIIB
rat
10 insulin
receptor
exon 11
human

11 NCAM,
exon MSDb
mouse

12 cTNT,
exon 5
chicken

13 AMP
deaminase 1
exon 2
rat

14 CD44,
exon 5
mouse

15 FGFR-1,
exon α
human

16 myosin
heavy chain,
exon 18
Droso-phila

17 FcγRIIA
exon Tm
human

18 interleukin-3α, exon 8 mouse

19 DHFR, exon 2A, reporter gene hamster

20 HIV-1,
exon 6D
HIV-1

21 SRp20, exon 4 human

22 PPT, exon 4 rat


B: minigenes containing multiple cassette exons
# name species minigene reference
23 fast sceletal TnT, exons 4-7 rat

24 CD45, exons 4-6 human, mouse

25 APP, exons 7,8 human


C: minigenes containing mutually exclusive exons
# name species minigene reference
26 α-tropo-myosin exons 2,3 rat

27 α-tropo-myosin exons NM,SK human

28 β-tropo-myosin exons 6A,B chicken

29 β-tropo-myosin exons 6,7 rat

30 pyruvate-kinase M exons 9,10 human

31 albumin, exons G,H rat

32 MLC, exons 1/3 rat

33 FGFR,
K-SAM
human


D: minigenes containing a retained intron
# name species minigene reference
34 bGH, intron D bovine


E: minigenes containing incremental combinatorial exons
# name species minigene reference
35 tau, exons 2,3 human


F: minigenes containing alternative 3' splice sites
# name species minigene reference
36 CT/CGRP human

37 dsx RO Droso-phila

38 M-tra Droso-phila

39 BPV-1 BPV-1


G: minigenes containing alternative 5' splice sites
# name species minigene reference
40 E1A Adeno-virus

41 SERCA2, exon 2a hamster

42 Caldesomon gene human

43 SWAP human

44 SC40
t-antigen
SV40

45 β-globin, β-thalassemic allele human


H: minigenes containing alternative 3'- and 5' splice sites
# name species minigene reference
46 fibronectin, exon IIICS human