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7.3 Pharmacological Mechanisms of Splicing Modulation 165
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7.3.1.7 Stabilization of Tr a n s -Regulatory Element RNA–Protein
Interactions (RPIs)
An alternative strategy to inhibition of splicing factor protein–protein interactions
is stabilization of splicing factor RNA–protein interactions (RPIs). Spliceosome
assembly is a dynamic process that requires not only the orderly association of
components for proper progression through the assembly steps but also their eventual dissociation. Cancers bearing mutations in splicing factor and spliceosomal
genes may be particularly vulnerable to association/dissociation imbalance as these
malignancies are wholly dependent on the remaining wild-type machinery to fulll
their pre-mRNA splicing needs [76]. Cognizant of this therapeutic connection,
Kielkopf et al. developed a uorescent polarization screening assay to identify
small-molecule stabilizers of a U2AF2/SF1/U2AF1
S34F
ternary complex with a
uorophore-labeled RNA containing a minimal 3′ss (Figure 7.10b) [77]. The choice
incorporation of mutant U2AF1
S34F
was based on the frequency of this mutation in
myelodysplastic syndromes and lung adenocarcinomas. Nearly 1500 compounds
from the NCI Developmental Therapeutics Program were screened and one hit was
identied – NSC194308 (EC50∼100 μM, Figure 7.10c), which was subject to in vitro
splicing validation in HeLa cells where it inhibited splicing of the AdML minigene
transcript (∼70% intron retention at 50 μM) and eliminated spliceosomal complexes B and C as expected for U2AF2/SF1/U2AF1–RNA interaction stabilization.
Furthermore, NSC194308 exhibited modest selectivity for K562 leukemia cells with
the U2AF1
S34F
mutation over wild-type U2AF1 (CC50= 5 μM vs. 18 μM, respectively), which was attributed to sequence specicity for canonical polypyrimidine
tracts – U2AF2’s cis-regulatory sequence – over degenerate tracts. Structural studies
concluded NSC194308 bound an open-form U2AF2 conformation in a pocket at the
interface of two RNA-interacting domains. Limited hit expansion established the
importance of hydrophobic groups occupying a protein pocket (NSC194308 pinane
and NSC187514 adamantane compared NSC194285 cyclohexane, Figure 7.10c) and
an anionic thiosulfate in the vicinity of the pre-mRNA (NSC194308 compared to
MAA). The amidine would be expectedly cationic and potentially interactive with
the negatively charged RNA phosphate backbone but was not scrutinized.
7.3.2 Kinases and Phosphatases
Kinases regulate all aspects of pre-mRNA splicing, including (i) splice site
recognition, (ii) spliceosome assembly, and (iii) spliceosome function. The most
prominent kinase families belong to the CDK-MAPK-GSK3-CLK (CMGC) group
and notably include CDC2-like kinases (CLKs), dual-specicity tyrosine-regulated
kinases (DYRKs), serine-arginine protein kinases (SRPKs), and Prp kinases. Many
nontraditional kinases have been implicated as well, such as topoisomerase I
[78]. The primary substrates of these kinases are splicing factors through which
most pre-mRNA splicing modulation is mediated. The rst FDA-approved drug
targeting a kinase was imatinib in 2001 (albeit not for splicing), since then, a
remarkable number of tools were established that expedite kinase drug discovery
and development [79]. However, despite the great advances in the eld, only
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one kinase inhibitor has entered clinical trials for which pre-mRNA splicing is a
claimed mechanistic feature – cirtuvivint (from Biosplice Therapeutics, formerly
Samumed), a pan-CLK/DYRK family inhibitor currently under Phase I anticancer
investigation (Figure 7.11a) [80, 81]. This clinical paucity is noteworthy considering
Examples of splicing modulating kinase inhibitors
N
N
N
pan-CLK/DYRK inhibitor
Topo I kinase inhibitor
F
NH
O
Cirtuvivint
HN
O
N
H
C77
CF
3
O
N
H
N
SRPIN340
SRPK1 inhibitor
N
N
N
N
H
O
Atuveciclib
CDK9 inhibitor
N
N
N
(a)
CLK1 autoregulation of CLK1 alternative splicing
Exon 3
5’ss 3’ss 5’ss 3’ss
HO
N
N
O O
S
F
O
S
NH
Exon 4 Exon 5
S
16
CLK1/DYRK1A inhibitor
H
O
N
O
N
SB216763
GSK3 inhibitor
O
Et
SRPKIN-1
SRPK1/2 inhibitor
O
N
N
H
Intron 4
O
OH
Cl
Cl
N
H
O
THZ1
CDK7 inhibitor
CN
N
H
H
N
O
Harmine
DYRK1A inhibitor
N
N
N
O
LY2090314
GSK3 inhibitor
Cl
O
N
H
Cl
O
AT7519
CDK9 inhibitor
Cl
N
N
N
H
NH
N
N
O
N
NH
O
F
NH
N
NH
N
H
Exon 3
mRNA degraded
(b)
Figure 7.11 (a) Examples of kinase inhibitors that modulate splicing. (b) CLK1
autoregulation of CLK1 pre-mRNA alternative splicing. Exon 4 skipped isoform is rapidly
degraded, whereas the intron 4 detained isoform is stable but cannot leave the nucleus and
cannot be translated.
Exon 5
Exon 3
mRNA stable but not translated
Exon 3
mRNA encodes functional CLK1
Exon 4
Exon 4
Exon 5
Detained
intron 4
Exon 5
7.3 Pharmacological Mechanisms of Splicing Modulation 167
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the huge number of splicing modulating kinase inhibitors discovered to-date, but,
as it turns out, kinase regulatory biology presents certain development challenges.
7.3.2.1 Challenges in Targeting Kinases
Leaving aside the traditional issue of kinase specicity, the rst challenge to
modulating splicing through kinases is substrate selectivity. Although splicing factors are major targets of phosphorylation by CMGC kinases, they are not
the only ones. For example, CLK1 – perhaps the most studied splicing-relevant
kinase – regulates 5′ss recognition through phosphorylation of SRSFs. Incredibly,
it also regulates 5′ss recognition through U1 snRNP; recent evidence suggests that
the U1-70K protein component of U1 must be phosphorylated by CLK1 to trigger
pre-mRNA recruitment and integration with SRSFs already present at the 5′ss [82].
Second, CMGC kinases are highly redundant, although the extent to which each
can compensate for the loss of another is still a topic of active research. In 2014,
Engel et al. presented evidence that DYRK1A can compensate for CLK1 inhibition
to moderate splicing ecacy, necessitating the development of dual CLK1/DYRK1A
inhibitors to achieve pre-mRNA splicing modulation [83]. Substrate overlap is probably not perfect, however. SRPK1 and SRPK2 have been reported to act at dierent
spliceosome assembly steps [84], and Cochrane et al. have demonstrated that CLK1
and CLK2 regulate distinct steps and exhibit opposing roles in HIV-1 expression [85].
A third challenge concerns the poorly understood interactome of these kinases.
Studies on cancer-relevant kinases have revealed regulatory roles for nonsubstrate
proteins that modify catalytic activity [86–88]. Perhaps the most striking example
of this in the context of splicing is the relationship between CLK1 and SRPK1.
Although SRPK1 is broadly distributed throughout the cell, CLK1 is localized
entirely to the nucleus where it maintains the SRPK1 nuclear pool via formation
of a stable equimolar complex [89]. The aliation is reciprocal; SRPK1 stimulates
release of CLK1 from its phosphorylated SRSF substrates – a critical step that
permits unobstructed binding of U1 snRNP to the 5′ss.
A fourth challenge in the development of kinase modulators is autoregulation.
CLKs [90], DYRKS [91], and SRPKS [92] all self-regulate their catalytic activity via
autophosphorylation, although not always through the same mechanism. DYRK
self-phosphorylation, for example, is restricted to translation just prior to ribosomal
release, whereas CLKs and SRPKs can phosphorylate themselves at any time [93].
More remarkable is the ability of these kinases to autoregulate their own pre-mRNA
splicing. For instance, catalytically competent full-length CLK1 contains all 13
CLK1 exons, but alternative splicing aords two inactive isoform options: (i) an
exon 4 skipped transcript that is rapidly degraded through nonsense-mediated
decay (NMD) and (ii) a stable intron 4 detention transcript [94, 95]. Under normal
conditions, CLK1 suppresses its own ecient splicing and detained-intron CLK1
transcripts pool in the nucleus. However, to compensate for small-molecule blockade, CLK1 promotes excision of intron 4 and upregulates formation of full-length
CLK1 mRNA (Figure 7.11b).
Finally, RNA transcription and pre-mRNA splicing are intrinsically intertwined
and dicult to selectively modulate because both processes (i) occur contemporaneously and (ii) are regulated by CMGC family kinases [96]. In fact, the RNA
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polymerase II (RNAP II) transcription complex recruits splicing factors to the elongating pre-mRNA transcript to initiate splicing before transcription is even complete
[97, 98]. Furthermore, RNAP II activity is regulated by cyclin-dependent kinases
(CDKs), which are CMGC family members and closely related to splicing kinases
[96]. It should be noted that some CDKs perform double duty; for example, proper
interaction between U2 snRNP and the U5/U6 snRNPs requires phosphorylation of
SF3B1 by CDK11 [99].
7.3.2.2 Inhibition of Kinases
Perhaps the most eective strategy in the discovery of kinase inhibitors is focused
library screening [100, 101]. In contrast to traditional library design schemes – which
favor broad chemical diversity for maximum generality – focused libraries are tailored to specic biological targets. Two classes are recognized: (i) ligand-based
libraries use known compounds with desirable biological activity as templates and
(ii) structure-based libraries utilize structural and physicochemical properties of the
biological target to inform compound selection.
Despite the maturity of the kinase drug development eld, the design of highly
specic single-kinase inhibitors remains nearly impossible as compounds invariably possess unintended activity with unexpected kinases [102]. However, those
unexpected kinases possibly include those which regulate pre-mRNA splicing, and
it is this consideration that underlies the design of ligand-based focused libraries
constructed from known kinase inhibitors. Webb et al. screened 2035 characterized
kinase inhibitors for promoters of exon skipping in a whole-cell, MDM2-based,
luciferase reporter model and identied hits milciclib, PF-3758309,andPF-562271
(Figure 7.12) [102, 103]. Ostensibly, these compounds were CDK, PAK, and FAK
inhibitors, respectively, but closer examination discovered all three to be inhibitors
of splicing-associated CLK-family kinases. As expected, these compounds reduced
phosphorylation of splicing factor SR proteins. Interestingly, PF-562271 also
suppressed phosphorylation of SF3B1, a known substrate of both DYRK1A and
CDK-family kinases; however, PF-562271 does not inhibit these, suggesting SF3B1
may be regulated by another as-yet unknown kinase.
7.3.2.3 Activation and Degradation of Kinases
The relative lack of success with kinase inhibition urges investigation into unconventional mechanisms of kinase modulation – agonism and degradation. Although
N
N
Developed as a CDK inhibitor
Figure 7.12 (a) Kinase inhibitors with previously unrecognized splicing activity discovered
through ligand-focused library screening.
H
N
N
N
Milciclib
CLK inhibitor
NH
H
N
N
N
O
N
HN
S
N
N
PF3758309
CLK inhibitor
Developed as a PAK inhibitor
H
N
N
N
O
O
N
H
PF562271
CLK inhibitor
Developed as a FAK inhibitor
H
N
N
N F
OSO
N N
H
N
F
F
7.3 Pharmacological Mechanisms of Splicing Modulation 169
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the following small molecules represent novel approaches to modulating splicing,
both were identied serendipitously. Unfortunately, principles in the deliberate discovery of kinase agonists and degraders remain underdeveloped; until they mature,
these mechanisms will be dicult to advance. Moreover, the advantages of these
mechanisms over traditional kinase inhibition have not been methodically assessed.
In 2015, a dual-reporter minigene-based high-throughput screen from Hagiwara
et al. discovered a small-molecule kinase activator that rescued inclusion of ELP1
exon 20 (RECTAS, Figure 7.13a) [104]. Aberrant ELP1 splicing is hallmark of familial dysautonomia: a single U-to-C mutation in the +6 position of intron 20 induces
exon 20 skipping, the protein product of which is dysfunctional (Figure 7.13b).
Several observations established RECTAS’ mechanism of action: (i) intron
20 contained several ISE cis-regulatory binding sites for SRSF6, (ii) pulldown
experiments identied CLK1 as RECTAS’ biological target, (iii) SRSF6 phosphorylation/activation was enhanced by RECTAS, and (4) SRSF6 is a known substrate
of CLK1. Furthermore, CLK inhibitors induced the opposite eect; suppression of
SRSF6 phosphorylation and greater exon 20 skipping [105].
Another unusual kinase modulation mechanism is protein degradation. Expressly
interested in discovering novel DYRK1A inhibitors, Hagiwara et al. developed a
cell-based screening assay and identied CaNDY (Figure 7.13a), which was initially
believed to be a classic ATP-competitive kinase inhibitor [106]. However,expression
studies revealed selective depletion of DYRKs and CLKs (but no other kinases)
reversible upon co-treatment with proteasome inhibitors. These results suggested
that CaNDY functioned as a selective degrader of DYRKs and CLKs, and the
compound was advanced for evaluation in splicing-associated cystic brosis. The
origin of the disease in 60% of Class V cystic brosis patients can be traced to an
′
intronic C-to-U mutation in CFTR that generates a false 5
ss that – when paired with
a 3′ss-like sequence upstream – leads to pseudoexon inclusion (Figure 7.13c) [107].
The pseudo-3′ss is regulated by exonic SRSFs that enhance an otherwise weak
U2 snRNP interaction, but the SRSFs are themselves regulated by CLK kinases;
consequently, degradation of CLKs by CaNDY promoted pseudoexon skipping and
restored functional CFTR expression.
7.3.2.4 Inhibition and Activation of Protein Phosphatases
Phosphatases perform regulatory roles complementary to those of kinases: where
kinases phosphorylate splicing factors and activate them toward pre-mRNA binding,
phosphatases dephosphorylate splicing factors and deactivate them [108, 109]. Additional substrates include the SF3B2 protein component of U2 snRNP, which must
be dephosphorylated by protein phosphatase 2C (PP2C) to facilitate spliceosomal
assembly of Complex A [110, 111]. It is therefore unsurprising that phosphatases
were of great interest in the early history of splicing modulation by small molecules;
by 1992, there were more examples of phosphatase inhibitors than there were of
kinase inhibitors (although these were predominantly structurally complex secondary metabolites, Figure 7.14a) [13]. However, interest in phosphatases withered
during the kinase drug development explosion and has not since recovered. To date,
the only noteworthy discovery was that of homoharringtonine (Figure 7.14a),
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Small molecule kinase activators and degraders
O
NH
N
N
Cl
RECTAS
CLK activator
(a)
Familial Dysautonomia – ELP1 exon 20 skipping
Exon 19 Exon 20 Exon 21
5’ss 3’ss
SRSF6
O
H
N
N
O
Selective DYRK/CLK degrader
+6 U
CaNDY
C mutation
S
SRSF6
SRSF6
NH
NH
P
P
Exon 19
Translated to dysfunctional IKAP protein Translated to functional IKAP protein
Exon 20Exon 21Exon 19
Exon 21
(b)
Cystic Fibrosis – CFTR pseudoexon inclusion
P
C
SRSF
Exon 23
SRSF
Exon 23
mRNA degraded or translated into defective protein
Psi23
U2
5’ss 3’sspsi-3’ss psi-5’ss
P
Psi23
U mutation
Exon 24
SRSF
Exon 24Exon 24 Exon 23
Translated to functional protein
(c)
Figure 7.13 (a) Kinase activator RECTAS and kinase degrader CaNDY. (b) ELP1 exon 20 is
skipped due to a U-to-C intronic mutation, resulting in dysfunctional IKAP protein and
familial dysautonomia; RECTAS enhanced SRSF6 phosphorylation and promoted exon
20 inclusion. (c) A C-to-U intronic mutation in CFTR leads to pseudoexon inclusion and loss
of functional protein; CaNDY degraded CLK kinases responsible for SRSF
phosphorylation/activation and suppressed pseudoexon inclusion.
an anticancer drug approved in 2012 under the tradename omacetaxine. A 2018
mechanistic investigation by Lu et al. uncovered a splicing connection through
which homoharringtonine promoted alternative 5′ss usage favoring formation of
the proapoptotic short Bcl-xS transcript over the tumorigenic long Bcl-xL transcript
(Figure 7.14b) [112, 113]. Overexpression, knockdown, and chemical genomic
studies implicated phosphatase PP1 activation (not inhibition) as the primary
mediator of homoharringtonine’s splicing activity.
7.3 Pharmacological Mechanisms of Splicing Modulation 171
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Examples of splicing modulating phosphatase inhibitors and activators
O
O
O
OH OH OH
O
O
O
O
Tautomycin
PP1 and PP2A inhibitor
H
O
O
O
OH
H
OH
O
O
H
OH
O
PP1 and PP2A inhibitor
OH
O
O
N
N
H
N
OH
HO
O
N
O
O
P
O
OH
O
O
OHOH
Calyculin A
PP1 inhibitor
O
O
Homoharringtonine
PP1 and PP2A activator
(a)
Alternative 5’-splicing of BCLX pre-mRNA
O
H
Okadaic acid
O
OH
O
O
OH
O
O
H
O
alt 5’ss
OH
H
O
O
H
HO
O
N
H
NH
NH
N
O
O
OH
H2N
O
NH
N
O
O
N
H
O
HN
O
Microcystin-LR
PP1 and PP2A inhibitor
OH
N
O
2
O
O
HN
OH
Translated to anti-apoptotic Bcl-xL
(b)
Figure 7.14 (a) Examples of phosphatase inhibitors and activators that modulate
pre-mRNA splicing. (b) Illustration of BCLX alternative 5′-splicing. Use of an alternative 5′ss
truncates exon 2, afforded short transcript encoding the proapoptosis factor Bcl-xS.
Exon 1
5’ss 3’ss 5’ss 3’ss
Exon 2a 2b
+ Small molecule
Exon 3
2bExon 2aExon 1
Exon 1
Translated to pro-apoptotic Bcl-xS
Exon 3
Exon 2a
Exon 3
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7.3.3 Epigenetic Writers and Erasers
Epigenetic tools – readers, writers, and erasers – regulate gene transcription through
the post-translational modication of histones by either transferring (i.e. writing),
removing (i.e. erasing), or recognizing (i.e. reading) so-called “marks,” of which the
most common marks are acetyl and methyl groups [114]. Because epigenetic tools
control transcription, and transcription is contemporaneous with pre-mRNA splicing, small-molecule targeting of epigenetic tools may lead to splicing modulation.
However, as with many previous examples, the regulatory roles of epigenetic tools
are complex, and the distinction is blurred between transcriptional control and
splicing control. For instance, some RNA-binding proteins are directly regulated
by epigenetic writers; the acetyltransferase CBP directly acetylates splicing factor
Sam68, in addition to histones [115]. U6 snRNP activity is also dependent on
writers; adenosine 43 of the U6 snRNA must be N-methylated by methyltransferase
METTL16 for 5′ss recognition [116, 117]. Nevertheless, the rst epigenetic tools
targeted by small-molecule splicing modulators were histone deacetylases (HDACs)
as inhibition arrests spliceosome assembly [118–121]. Structurally, HDACinhibitors
(e.g. SAHA, LBH589, and M344; Figure 7.15) are recognizable by their shared
hydroxamic acid functionality, a key pharmacophore element that coordinates and
inactivates a catalytically required metal ion – usually zinc – in the HDAC active site
[122]. Histone acetyl transferase (HAT) inhibitors, such as anacardic acid, aect
splicing as well; in some cases, the molecular mechanism of inhibition involves
catalytically noncompetitive occlusion of substrate binding [123, 124].
7.3.3.1 Inhibition of Epigenetic Writers
Methyl transferase inhibitors also modulate splicing – the rst of which was
RG3039, an inhibitor of DcpS discovered in 2005 [121, 125, 126] – and today receive
greater attention than HDACs and HATs. The most popular methyl transferase is
PRMT5, an oncogene whose dysregulation leads to epigenetic inactivation of tumor
suppressor expression and promotion of androgen receptor expression [127, 128];
however, PRMT5 has additional roles, including an obligation to methylate spliceosomal Sm proteins for proper snRNP assembly [129–131]. These dual duties make
PRMT5 inhibition an attractive target for the treatment of splicing-addicted cancers,
which are dependent on protumorigenic alternatively spliced isoforms arising from
dysregulated splicing.
In 2021, Janssen reported the structure-based discovery of the PRMT5 inhibitor
onametostat (JNJ-64619178, Figure 7.16) [132]. PRMT5 requires SAM as a
methyl source, and it was reasoned that adenosine-based small molecules might
compete with SAM and thus antagonize transferase activity. The company rst
constructed a focused chemical library composed of small molecules whose design
was based on Eli Lilly’s 2012 ternary co-crystal structure of PRMT5 in complex with
(i) MEP50 – an adaptor protein, (ii) a peptide model of a histone substrate, and (iii) a
natural inhibitor (A9145C) [133]. The focused library was screened in a biochemical
assay for inhibition of SAM consumption, and hits were triaged in a whole-cell
assay monitoring methylation of spliceosomal Sm proteins [134]. Hit expansion
HN
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Cl
Cl
N
H
LBH589
HDAC inhibitor
N
O
RG3039
DcpS inhibitor
N
NH
7.3 Pharmacological Mechanisms of Splicing Modulation 173
H
N
OH
O
N
H
N
O
H
N
OH
O
M344
HDAC inhibitor
H
N
O
O
OH
N
H
SAHA
HDAC inhibitor
NH
2
N
2
OOH
OH
Anacardic acid
HAT inhibitor
H
N
O
F
HO
F
F
N
OH
PF06939999
Selective PRMT5 inhibitor
N
H
N
N
N
HO
O
AGI24512
Indirect PRMT5 inhibitor
O
N
N
O
O
HO
O
BIX01338
HMT inhibitor
O
MS023
PRMT1 inhibitor
O
N
NH
N
NH
N
NH
CF
3
2
Figure 7.15 Examples of epigenetic tool inhibitors. The key pharmacophore feature of
HDAC inhibitors (hydroxamic acid, top row) is colored blue.
underscored the critical and stereo-discriminating inuence of a basic amine – a
structural feature that, for undisclosed reasons, was abandoned during lead optimization. Onametostat is a potent Sm methylation inhibitor compared to its early
analogs (IC
cell
= 0.2 nM; analogs in Figure 7.16), but long assay incubation times
50
are required for complete reduction of methylated Sm proteins (original = 48 hours,
long = 96 hours), suggesting that prolonged and continuous exposures are necessary for maximum eect. An explanation for this time-dependent phenomenon
came from kinetic studies, which indicated a very long PRMT5 residence time
for onametostat. From a splicing perspective, RNA-seq showed greater splicing
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N
H2N
HO
O
O
+
S
N
N
OHHO
S-adenosyl methionine (SAM)
PRMT5 co-substrate
Br
H2N
H2N
HN
N
HO
PRMT5
IC
50
Sm-Me
IC
50
N
HO
PRMT5
IC
50
Sm-Me
IC
50
N
O
N
N
OH
4
= 52,000 nM
= 2,300 nM
O
N
OH
13
= 83 nM
= 2,400 nM
NH
2
N
O
N
HO
Onametostat
Selective PRMT5 inhibitor
PRMT5
IC
NH
N
N
N
N
= 0.1 nM
50
Sm-Me
IC
= 0.2 nM
50
2
NH
2
N
NH
HO
O
N
OH
N
2
H2N
H2N
O
A9145C
General methyltransferase inhibitor
NH
N
OH
HN
2
N
N
N
IC
50
IC
50
IC
N
PRMT5
Sm-Me
N
50
IC
50
HO
HO
PRMT5
Sm-Me
O
OH
16
= >10,000 nM
= >10,000 nM
O
OH
14
= 9.5 nM
= 58 nM
NH
N
N
N
2
N
NH
2
N
N
NH
2
N
Figure 7.16 PRMT5 inhibitor onametostat, PRMT5 cosubstrate SAM, natural secondary
metabolite A914C, and early onametostat analogs. IC
PRMT5 catalytic inhibition; IC
spliceosome Sm proteins.
disruption in liquid tumor lines sensitive to PRMT5 inhibition than in insensitive
lines, with a signicant increase in exon skipping, intron retention, and alternative
splice site usage; however, no such correlation was observed in solid tumor lines
carrying splicing factor mutations. Nonetheless, onametostat entered Phase I trials
in 2018 for both cancer types, but patient enrollment was discontinued in 2021 due
to disappointing clinical endpoints despite demonstration of robust PRMT5 target
engagement [135].
7.3.4 RNA Helicases
RNA helicases regulate pre-mRNA splicing by remodeling pre-mRNA cis-regulatory
structures and therefore represent plausible drug discovery targets; some special
cases are also essential for proper spliceosome assembly [136–139]. However, this
PRMT5
is an enzymatic assessment of
Sm-Me
is a whole cell assay measuring methylation of
50
50
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