01/02.2020 | THE SCIENTIST 43Therefore, mis-splicing has a strong potential to be implicated in
disease. Parkinson’s, progeria, cardiomyopathy, spinal muscular
atrophy, myotonic dystrophy, breast cancer, ovarian cancer—these
devastating illnesses and many more appear to be caused partly
by defects in RNA splicing, emphasizing the range of crippling
effects that can stem from even slightly tipping the balance of
protein isoform expression.One scenario involves the titin (TTN) gene, which holds the
record for the highest number of exons—a whopping 363—among
all mammalian genes and encodes the largest known protein in the
human body, weighing in at 4.2 megadaltons. The TTN protein is a
molecular spring that contributes to the elasticity of heart muscle.
Over the course of cardiac development, there is a gradual increase
in the frequency of TTN exon skipping by the spliceosome, and
these exons are thus spliced out from the mRNA. (See illustration
on opposite page.) The transition from the fetal to the adult cardiac
titin isoform is part of the normal developmental program and is
orchestrated by an RBP called RBM20. That RBP promotes skip-
ping where it’s found, thus inducing a shift in protein expression
from a long, elastic TTN isoform to a short, stiff isoform. Using a
rodent model, an international cohort of researchers and physi-
cians demonstrated that the absence of RBM20 causes TTN mis-
splicing, leading to the buildup of long, elastic TTN and phenotypes
resembling the decreased heart contractility seen in humans with
dilated cardiomyopathy induced by mutations in RBM20.^16 The
results strongly suggest that TTN mis-splicing contributes to
RBM20-linked cardiomyopathy.
Another example is DMD, the gene that encodes the dystrophin
protein, which is important for muscle integrity and force transmis-
sion. Mutational variants in DMD are notoriously associated with
Duchenne muscular dystrophy, a disease that severely impairs mus-
cle function. (See “Mending Muscle,” September 2018.) One disease-
causing DMD mutation is a multiexon deletion that commonly
results in a frameshift starting at exon 51. Splicing the remaining
exons together results in a shortened dystrophin protein with com-
promised function. The lack of fully functioning dystrophin protein
causes muscle weakness and atrophy, which drastically limit the
physical abilities of people suffering from this disease.
Some therapies currently in development for Duchenne mus-
cular dystrophy and other diseases aim to correct defects in splicing
to alleviate symptoms. For example, researchers have been able
to partially recover dystrophin function by using antisense oligo-
nucleotides that prevent the spliceosome from recognizing exons
downstream of the deletion. By hiding these regions from the spli-
ceosome, the exons will be skipped. This can then restore the read-
ing frame and produce a near-full-length protein. Tw o years ago,
scientists at Japan’s National Center of Neurology and Psychiatryreported results from a Phase 1 trial hinting at the oligonucleotides’
safety and capacity to induce DMD exon-skipping in patients with
Duchenne muscular dystrophy.^17
Understanding the story behind each protein in our bodies
has turned out to be far more complex than reading our DNA.
Although the basic splicing mechanism was uncovered more
than 40 years ago, working out the interplay between splicing
and physiology continues to fascinate us. We hope that advanced
knowledge of how alternative splicing is regulated and the
functional role of each protein isoform during development
and disease will lay the groundwork for the success of future
translational therapies. gGabrielle M. Gentile, Hannah J. Wiedner, and Emma R. Hinkle are
graduate students in the Curriculum in Genetics and Molecular
Biology at the University of North Carolina at Chapel Hill, where
Jimena Giudice is an assistant professor in the Department of Cell
Biology and Physiology. Giudice is also a member of the university’s
McAllister Heart Institute in the School of Medicine.References- S.M. Berget et al., “Spliced segments at the 5' terminus of adenovirus 2 late mRNA,”
PNAS, 74:3171–75, 1977. - L .T. Chow et al., “A n amazing sequence arrangement at the 5' ends of adenovirus 2
messenger R N A ,” Cell, 12:1–8, 1977. - W. Gilbert, “Why genes in pieces?” Nature, 271:501, 1978.
- R.T. Boggs et al., “Regulation of sexual differentiation in D. melanogaster via
alternative splicing of RNA from the transformer gene,” Cell, 50:739–47, 1987. - L.R. Bell et al., “Sex-lethal, a Drosophila sex determination switch gene, exhibits
sex-specific RNA splicing and sequence similarity to RNA binding proteins,” Cell,
55:1037–46, 1988. - B.S. Baker, M.F. Wolfner, “A molecular analysis of doublesex, a bifunctional
gene that controls both male and female sexual differentiation in Drosophila
melanogaster,” Genes Dev, 2:477–89, 1988. - K.C. Burtis, B.S. Baker, “Drosophila doublesex gene controls somatic sexual
differentiation by producing alternatively spliced mRNAs encoding related sex-
specific polypeptides,” Cell, 56:997–1010, 1989. - Q. Pan et al., “Deep surveying of alternative splicing complexity in the human
transcriptome by high-throughput sequencing,” Nat Genet, 40:1413–15, 2008. - J.C. Castle et al., “Expression of 24,426 human alternative splicing events and
predicted cis regulation in 48 tissues and cell lines,” Nat Genet, 40:1416–25, 2008. - E.T. Wang et al., “Alternative isoform regulation in human tissue transcriptomes,”
Nature, 456:470–76, 2008. - J. Merkin et al., “Evolutionary dynamics of gene and isoform regulation in
mammalian tissues,” Science 338:1593–99, 2012. - J. Giudice et al., “Alternative splicing regulates vesicular trafficking genes in
cardiomyocytes during postnatal heart development,” Nat Commun, 5:3603, 2014. - E.T. Wang et al., “Antagonistic regulation of mRNA expression and splicing by CELF
and MBNL proteins,” Genome Res, 25:858–71, 2015. - F. Carrillo Oesterreich et al., “Splicing of nascent RNA coincides with intron exit
from RNA polymerase II,” Cell, 165:372–81, 2016. - M.M. Maslon et al., “A slow transcription rate causes embryonic lethality and
perturbs kinetic coupling of neuronal genes,” EMBO J, 38:e101244, 2019. - W. Guo et al., “RBM20, a gene for hereditary cardiomyopathy, regulates titin
splicing,” Nat Med, 18:766–73, 2012. - H. Komaki et al., “Systemic administration of the antisense oligonucleotide NS-065/
NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy,”
Sci Transl Med, 10:eaan0713, 2018.
Many devastating illnesses appear to be
caused partly by defects in RNA splicing.