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本书版权归John Wiley & Sons Inc.所有
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7
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Pre-mRNA Splicing Modulation
Scott J. Barraza and Matthew G. Woll
Chemistry Department, PTC Therapeutics, Inc., South Plainfield, NJ, USA
7.1 Introduction
One of the most startling surprises of the Human Genome Project was the discovery
that humans possessed fewer genes than was predicted; until 2001, estimates had
placed the number between 40,000 and 100,000 genes – roughly consistent with
the number of proteins – but the actual value appeared to be ∼30,000! [1, 2] Yet,
even this was too high, and has since been downwardly revised to a total of ∼19,000
genes [3] – a value lower than many “simple” animals and well below that of
plants [4]. The question of how so few genes could encode so many proteins had an
answer, however. The mechanism underlying protein diversity had been reported
as early as 1977 from pioneering research in the laboratories of Richard Roberts
(then at Cold Spring Harbor Laboratory) and Phillip Sharp (at the Massachusetts
Institute of Technology), for which both received the 1993 Nobel Prize in Physiology or Medicine [5]. Originally working with virus models – and later higher
organisms – the researchers revealed that genes were segmented and could encode
many protein variations through inclusion or exclusion of dierent segments at
the pre-mRNA level. This surprising diversity-generating mechanism was termed
“alternative splicing”. From the perspective of drug development, pre-mRNA
alternative splicing presents an attractive approach to the pharmacological intervention of human disease and may oer an eective solution to indirectly drugging
proteins traditionally considered “undruggable” through direct manipulation of
their pre-mRNA transcripts [6, 7].
Pharmacological modulation of alternative splicing was rst demonstrated with
antisense oligonucleotides (ASOs) in the 1980s [8–10], years before the landmark
publications from the human genome project. This feat would eventually culminate in the 2016 FDA approval of the rst splicing-modifying drug, nusinersen
(Spinraza®), for the treatment of spinal muscular atrophy (SMA) [11]. Though
trailing, small molecules were not far behind. The rst small-molecule splicing
modulators were kinase and phosphatase inhibitors reported in the early 1990s;
since then, many classes and mechanisms of small molecules have been discovered
151
RNA as a Drug Target: The Next Frontier for Medicinal Chemistry, First Edition.
Edited by John Schneekloth and Martin Pettersson.
© 2024 WILEY-VCH GmbH. Published 2024 by WILEY-VCH GmbH.
152 7 Pre-mRNA Splicing Modulation
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[12, 13]. Most successful of these is risdiplam (Evrysdi®), the rst small molecule
FDA-approved splicing-modulating drug, approved for use in SMA in 2020.
This chapter seeks to illustrate the mechanisms of pre-mRNA alternative splicing and highlight the various points in which small molecules may intervene with
curated examples. Larger molecules, such as oligonucleotides, will not be covered in
depth. Finally, several drug discovery case studies will be discussed – including that
of H3B-8800 and risdiplam – that will integrate splicing modulator development
with lessons from other chapters in this book.
7.2 Overview of Splicing Biology
7.2.1 The Spliceosome
The complex responsible for pre-mRNA splicing is the spliceosome, which is composed of six core snRNPs (small nuclear ribonucleoproteins) (Figure 7.1), each containing an snRNA (small nuclear RNA) and a dened set of proteins. Two types of
spliceosomes are recognized in humans: (i) the major spliceosome and (ii) the minor
spliceosome, which diers from the former in protein and RNA identity [14–16].
Furthermore, the major spliceosome resides solely in the nucleus, whereas cellular localization of the minor spliceosome remains contentious [17, 18]. This chapter
will focus on the major spliceosome as it is responsible for >99% of all pre-mRNA
splicing events [19]; however, it is worth noting that the minor spliceosome may be
a valid therapeutic target in a small set of diseases [20]. There also exist a handful of
protein-catalyzed splicing processes, such as the IRE1-mediated splicing of the XBP1
transcript [21], but these are rare regulatory mechanisms acting on transcripts that
have already been mostly processed by the major spliceosome. These special cases
will not be covered here.
The major spliceosome does not exist as a discrete entity in the nucleus; instead, it
is assembled in precise locations along the pre-mRNA transcript in a stepwise manner (Figure 7.2). It is important to note that the illustrated overview is characteristic
of “intron denition,” in which the growing spliceosome spans the intron to identify the exon-intron borders; this form of denition predominates when introns are
shorter than anking exons and is particularly common in lower eukaryotes (among
which yeast splicing has been extensively studied). In humans, however, introns
tend to be substantially longer than exons and the major mode of splice site recognition is consequently “exon denition” in which the spliceosome spans the exon from
the 3′ss of one intron to the 5′ss of the next. Because many details of exon denition
remain unsettled, and because it adds a great deal of complexity, it is omitted from
the splicing overview.
In the rst step of assembly, U1 snRNP “denes” the 5′-splice site (5′ss) – the
border that distinguishes the 3′end of one exon from the 5′end of the adjacent
intron – by binding a nucleotide sequence unique to the 5′ss. The result is complex
E and is followed by ATP-dependent formation of complex A, in which U2 snRNP
binds and denes the 3′-splice site (3′ss). In the subsequent and pinnacle step
of spliceosome assembly, U4/U5/U6 tri-snRNP is recruited to the 5′-ss to form
7.2 Overview of Splicing Biology 153
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Figure 7. 1 Core snRNPs (small nuclear ribonucleoproteins) of the human major
spliceosome. Each snRNP is composed of an snRNA (small nuclear RNA) and a defined set of
proteins.
precatalytic complex B – the system is ready to enter the catalysis phase. Transition
to the catalytically competent intermediate complex B* requires ATP-dependent
structural rearrangement followed by dissociation of U1 and U4 snRNP, after which
complex B* catalyzes intron-exon cleavage of the 5′ss and fusion to the 3′ss, forming
an RNA lariat-containing intermediate called complex C. The complex subsequently
severs the 3′ss, joins the exons, and releases the lariat, after which the spliceosome
is disassembled for additional cycles of pre-mRNA splicing.
154 7 Pre-mRNA Splicing Modulation
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5'
5'
5'
Complex E
Exon A
Exon A
Exon A
Complex A
pre-mRNA
SRSF
P
U1
Prp5 (+ATP)
Sub2 (+ATP)
5'
3'
Complex B
Intron
hnRNP
P
SRSF
P
Exon A
Exon B
Prp28 (+ATP)
hnRNP
P
5'
3'
3'SS5'SS
Exon B
Exon B
U1
U2
Exon A
Exon B
Exon B
3'
3'
U1
3'
Spliceosome assembly
U4
U6
U1
U5
U2
U2
and catalysis
U4
Brr2 (+ATP)
Snu114 (+ATP)
Prp2 (+ATP)
5'
U6
U5
5'
Exon A
5'
U1
5'
Exon A
3'
Exon B
Exon A Exon B
Prp22 (+ATP)
Exon B
U5
U5
U2
Exon A
Exon B
'
3
U6
U5
U2
mRNA
U6
Complex B*
3'
Post-spliceosomal
complex
Prp22 (+ATP)
Prp16 (+ATP)
U5
U2
Complex C
3'
Figure 7. 2 Overview of spliceosome assembly and pre-mRNA splicing catalysis. For clarity,
the intron definition mode of splice site recognition is illustrated, and splicing factors have
been omitted after Complex E.
7.2.2 Classes of Alternative Splicing
There are two major types of pre-mRNA splicing. The rst – constitutive splicing – is
the default condition in which exons are ligated in their linear order following
sequential intron removal. The second – alternative splicing – encompasses divergent events and is of greatest interest to drug developers. Many varieties of have
been observed (Figure 7.3). Exon skipping is a common type in which the exon
is spliced out of the nal mRNA and usually occurs when splice site recognition
is disrupted, or spliceosome function is depressed. Mutually exclusive skipping is
related, in which if one exon is included the other must be skipped. Intron retention
is the inclusion of an intron in the nal transcript and, like exon skipping, is often
associated with disturbed splice site recognition. A special regulatory case of intron
retention called intron detention will be explored later, in which nuclear pools of
partially-matured mRNA containing a single detained intron are available for activation by intron excision in response to cellular stimuli [22]. Alternative 5′-splicing
often occurs when an intronic sequence resembling a 5′-splice site is recognized
instead of the consensus site at the canonical exon-intron junction. Similarly,
alternative 3′-splicing occurs when an intronic 3′-splice site-like sequence is
recognized. Pseudoexon (or cryptic exon) inclusion shares properties of alternative
5′- and 3′-splicing. In this case, the intronic pseudoexons contain most but not all
features characteristic of a true exon.
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