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Recent
progress in Dr. Adrian
Krainer's laboratory has led to development
of a novel strategy for controlling the splicing machinery using synthetic
molecules. These molecules could potentially be used as a targeted therapeutic
approach for treatment of human diseases caused by splicing defects.
A typical mammalian gene is composed of several relatively short exons
that are interrupted by much longer introns. The production of a mature
mRNA by splicing of a primary transcript requires the coordinate action
of five small nuclear RNAs (U1, U2, U4-U6) and more than 100 polypeptides.
These components act at cis-elements within each intron (5' splice site,
3' splice site and branch site) to chemically join the appropriate segments.
This process must accurately identify and include all exons necessary
to generate a full-length message that correctly codes the protein product.
In addition to the sites directly involved in the splicing reaction,
other signals are required for accurate or efficient splicing, especially
when weak or regulated splice sites are involved. One class of signals
is represented by exonic splicing enhancers (ESEs). ESEs act as binding
sites for serine/arginine–rich proteins (SR proteins), a family
of highly conserved splicing factors that act at multiple steps of the
pre-mRNA splicing pathway. Analogous to transcriptional enhancers, SR
proteins have a nucleic acid recognition domain that binds the ESE and
an activation domain that recruits the splicing machinery to the site.
Chimeric molecules were designed to emulate the function of SR proteins
in recognizing exonic splicing enhancers and promoting exon inclusion.
These small (~5kDa) synthetic molecules combine a minimal RS domain
peptide with an antisense moiety complementary to the defective mRNA.
The antisense moiety can be a PNA (peptide-nucleic acid, consisting
of a peptide-like backbone and the four standard nucleobases) allowing
tandem synthesis with the peptide moiety. The PNA moiety targets the
defective exon by Watson-Crick base-pairing, whereas the RS domain peptide
recruits splicing factors to drive spliceosome assembly at the splice
sites flanking the target exon.
This approach, dubbed ESSENCE (for Exon-Specific Splicing ENhancement
by small Chimeric Effectors), has been shown to modify splicing in test
cases. A nonsense mutation in the breast cancer gene, BRCA1, results
in skipping of exon 18. This exon was included in vitro using a BRCA-specific
PNA-RS ESSENCE effector. Splicing was also redirected successfully in
vitro in one of two nearly identical copies of the survival of motor
neuron (SMN) gene, for which lack of functional protein results in a
neurodegenerative disease, spinal muscular atrophy (SMA). Transcripts
from one of the genes, SMN2, typically exclude exon 7, resulting in
an unstable protein product. In SMA patients, the other gene, SMN1,
is inactivated by mutation. Thus, restoration of correct splicing in
SMN2 by a sequence-specific PNA-RS effector could potentially complement
the genetic defect.
At least 15% of point mutations that result in human disease cause incorrect
splicing, leading in turn to a defective or truncated protein, or to
instability and destruction of the mRNA. In principle, the ESSENCE technique
could be used as a therapeutic strategy to restore proper gene expression
in various diseases in which one or more mutant alleles result in exon
skipping. Current efforts are devoted to optimizing the exon-specific
activators and their delivery into cells for in vivo studies.
Key Publications:
REVIEW: Cartegni L, Chew SL,
Krainer AR. Listening to silence and understanding nonsense: exonic
mutations that affect splicing. Nat Rev Genet. 2002 Apr;3(4):285-98.
abstract
ESE's: Liu HX, Zhang M, Krainer
AR. Identification of functional exonic splicing enhancer motifs recognized
by individual SR proteins. Genes Dev. 1998 Jul 1;12(13):1998-2012.
abstract
ESEfinder: Cartegni L, Wang J,
Zhu Z, Zhang MQ, Krainer AR. ESEfinder: a web resource to identify exonic
splicing enhancers. Nucleic Acids Res. 2003 Jul 1;31(13):3568-71.
abstract
SR PROTEINS: Zhu J, Krainer AR.
Pre-mRNA splicing in the absence of an SR protein RS domain. Genes Dev.
2000 Dec 15;14(24):3166-78. abstract
ESSENCE: Cartegni L, Krainer
AR. Correction of disease-associated exon skipping by synthetic exon-specific
activators. Nat Struct Biol 2003 Feb;10(2):120-5. abstract
press
releases
BRCA1: Liu HX, Cartegni L, Zhang
MQ, Krainer AR. A mechanism for exon skipping caused by nonsense or
missense mutations in BRCA1 and other genes. Nat Genet 2001 Jan;27(1):55-8.
abstract
press releases
BRCA2: Fackenthal JD, Cartegni
L, Krainer AR, Olopade OI. BRCA2 T2722R is a deleterious allele that
causes exon skipping. Am J Hum Genet 2002 Sep;71(3):625-31.
abstract
SMN2: Cartegni L, Krainer AR.
Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2
causes spinal muscular atrophy in the absence of SMN1. Nat Genet 2002
Apr;30(4):377-84. abstract |
