The spliceosome is a large molecular machine that catalyzes pre-mRNA splicing. It is composed of five small nuclear ribonucleoproteins (snRNPs) and over 300 proteins. Mutations that affect splicing can cause diseases like Duchenne muscular dystrophy and myelodysplastic syndrome (MDS). In DMD, splice-switching oligonucleotides induce exon skipping to restore the reading frame. In MDS, mutations in splicing factors SF3B1 and U2AF1 promote aberrant splicing and contribute to pathogenesis. Alternative splicing of VEGF pre-mRNA produces pro-angiogenic and anti-angiogenic isoforms impacting cancer. Detection methods like minigene assays and RT-PCR analyze

1. Spliceosome
Advanced molecular biology
Ahmed Adel Abdallah
Islamic University of Gaza
28.12.2021

2. Overview.
• Spliceosome machinery
• DMD
• MDS
• VEGF
• Detection

3. INTRO.
• Human genes contain 9 exons and 8 introns per gene.
• High-throughput RNA sequencing and analysis revealed that 90–95% of human multi-
exon genes produce transcript variants through AS.
• Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein
isoforms from a single pre-mRNA, significantly enriching the proteomic diversity of
higher eukaryotic organisms.
• About 9% of all mutations reported in the Human Gene Mutation Database (HGMD) are
splicing mutations (18761/208368) (HGMD database, accessed on October 10, 2017).
• Splicing mutations occur in more than 50% of all patients with MDS, implicating
spliceosome dysfunction as a key driver of disease pathophysiology.

4. INTRO.
• Several mechanisms are responsible for the formation of protein isoforms, such as
genetic variations, somatic recombination, post-translational and proteolytic
modifications, and alternative splicing (AS).
• Genes are composed of introns and exons, but only exons contain the information
necessary to make proteins.
• AS process needs to be efficiently spatiotemporally coordinated to yield a mature mRNA
that is exported from the nucleus to the cytoplasm to be translated into protein.
• The mRNA processing accomplished in 3 steps:
• 5′ capping,
• 3′ cleavage/polyadenylation,
• And RNA splicing.

5. • RNA splicing is a nuclear process catalyzed by large macromolecular machineries,
composed of small RNAs and proteins.
• Five small nuclear ribonucleoproteins U1, U2, U4, U5, U6 (snRNPs) and multiple
proteins (>300) cooperate to form the spliceosome.
• These snRNPs are essential for orchestrating the splicing reaction, and also
participate in the formation of spliceosome complex.
• In most of cases (98.7%), the exon/intron boundary sequences contain GT and AG
motifs at the 5′ and 3′ ends of the intron, respectively.
• Noncanonical GC-AG and AT-AC sequences at the splice sites occur in 0.56 and 0.09%
of the splice site pairs.
The spliceosome machinery

6. The spliceosome machinery
• Recognition of intron/exon boundary is directed by essential cis-elements (pre-
mRNA sequence): donor (5′) and acceptor (3′) splice sites, branch point and
polypyrimidine tract (PPT).
• The assembly of spliceosome is further coordinated by auxiliary cis-elements:
intronic/exonic splicing enhancers (ISEs/ESEs) and intronic/exonic splicing silencers
(ISSs/ESSs).
• The splicing trans-factors for exonic splicing enhancers (ESEs) are serine/arginine
(SR)-rich proteins, while splicing silencer elements (ISSs/ESSs) are heterogeneous
nuclear ribonucleoproteins (hnRNPs).

7. The spliceosome machinery
• Splicing is accomplished in two steps: recognition of intron/exon boundary and
catalysis of the transesterification reaction to excise out an intron followed by joining
two exons.
• During the splicing process, four complexes b/w pre-mRNA & spliceosome are
formed.
• The initial splicing procedure begins with recognition of 5’ SS (donor) GU by U1
snRNP, and the branch point by splicing factor 1 protein (SF1), and the
polypyrimidine tract (PPT) as well as 3’ accepter site AG by U2AF heterodimer
(U2AF65 and U2AF35, respectively). This step, known as an early complex (E).
• Binding of the U1 snRNP to the 5′ splice site is mediated by SRSF1, but only when
the RS-domain of SRSF1 is hyper-phosphorylated by CLK1 and SRPK1.
• Then, the SF1 is displaced from the branch point by the U2 snRNP, and A complex is
formed.

8. The spliceosome machinery
• The interaction between the branch point and the U2 snRNP protein is stabilized by specific
RNA helicases, and this is a signal for the recruitment of U4/5/6 tri-snRNP and formation of
the B complex (precatalytic spliceosome).
• additional RNA helicases leads to change of spliceosome conformation that leads to the
release of U1 and U4 snRNPs, the interaction between U6 with U2 snRNP, and the formation
of a pre-mRNA loop and the C complex.
• U6/U2 catalyzes transesterification reactions resulting in the formation of the lariat, followed
by removal of the intron lariat. Then the ends of exon are joined forming the spliced RNA
product.
• Splicing factors such as serine/arginine-rich (SR) proteins (SRSF) and the splicing factor 3b
complex (SF3B) work in association with the splicing core complex to coordinate AS.
• The expression levels and binding affinities of the different splicing factors play a
stoichiometric role in determining the final isoform of the protein that is to be expressed.

9. The
scheme
of the
splicing
process.

10. Mutations
• It is estimated that up to 15% of all point mutations that result in human genetic
disease cause an RNA splicing defect.
• Mutations in cis or trans splicing regulators cause aberrant splicing patterns that
often result in diseases in humans
• Next-generation sequencing of the patients’ genome DNA revealed that SF3B1
(splicing factor 3b subunit 1), U2AF1 (U2 small nuclear RNA auxiliary
factor 1), SRSF2 (serine/arginine rich splicing factor 2), and ZRSR2 (zinc
finger RNA binding motif & serine/arginine rich 2) are the most
frequently mutated splicing factors.

11. Duchenne muscular dystrophy
• Duchenne muscular dystrophy (DMD), is a fatal X-linked recessive neuromuscular disorder,
which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children.
• This fatal degenerative condition is caused by an absence of dystrophin in striated muscle,
this disrupt the reading frame resulting in unstable truncated products.
• The major DMD deletion "hot spot" is found between exons 45 and 53.
• Splice-switching oligonucleotides (SSO)-induced skipping of exon 51 restores the reading
frame and allows translation of internally truncated but partially functional dystrophin.
• SSOs targeting different exons would allow reading frame correction of over 50% of
deletions and 22% of duplications reported in the Leiden DMD-mutation Database.
• For these patients, restoration of the reading frame via antisense oligonucleotide-mediated
exon skipping is a promising therapeutic approach.
• It is anticipated that this approach will convert lethal DMD to milder form, BMD.

12. A duchenne muscular dystrophy gene hot spot
mutation in dystrophin.
Splice switching oligonucleotides (SSOs)
induce exon skipping.
(A) DMD>
- Premature stop codon.
(B) SSO>BMD>
- Exon 51 skipping by block ESE.
- Reading frame restored
- Partly functional dystrophin
- Becker-like phenotype.

13. SF3B1 and MDS
• SF3B1 mutations are found in a variety of myeloid malignancies, with extremely high
recurrence (48%–57%) in MDS subtypes.
• Surprisingly, SF3B1 mutations are responsible for the ring sideroblast (RS) phenotype
in ∼98% of cases.
• SF3B1 mutations in MDS patients have a cluster as a ‘hot spot’ at 700th residue of
Lysine changed to Glutamine, which resides in HEAT domain repeats.
• The mutation causes cryptic branch point sequence recognition. This creates
premature termination codons in the mRNA, resulting in nonsense-mediated decay
(NMD).

14. SF3B1 and MDS
• Under normal conditions, SF1 binds to the canonical branch site and U2AF binds
polypyrimidine tract.
• U2 snRNP then recruits SUGP1 via the interaction of the SF3B1 HEAT domain.
• SF3B1 stabilizes U2 snRNP binding to the branch point sequence during early
stages of spliceosome assembly.
• SUGP1 in turn assists in localizing U2 snRNP to the vicinity of the canonical BP.
• The SUGP1 G-patch domain then associates with and activates an unknown RNA
helicase required for the displacement of SF1, allowing base pairing between the
branch site and U2 snRNA
• When SUGP1 is depleted or mutated in the G-patch domain, the coupling
between SF1/ U2AF2 binding and ATP hydrolysis is absent, such that SF1 is not
displaced and thus blocks access to the canonical BP. U2 snRNP is then forced to
utilize an unblocked upstream cryptic BP.

15. SF3B1 in
myelodysplastic
syndromes MDS

16. U2AF1 and MDS
• U2AF1 mutations are detected in 5%–15% of MDS and 5%– 17% of CMML, as well
as at lower rates in other hematological malignancies.
• U2AF1 (U2AF35) is a small subunit of the U2AF heterodimer that is responsible
for the recognition of AG dinucleotide in pre-mRNA 3’ splice sites.
• The MDS mutations in U2AF1 alter RNA splicing and promote mis-splicing of
genes in ways that presumably contribute to abnormal hematopoiesis.

17. VEGF
• Vascular endothelial growth factor A VEGF 165b (anti-angiogenic) and VEGF165 (pro-
angiogenic) are generated from the same transcript, and their relative amounts are
dependent on alternative splicing.
• VEGF165b inhibits VEGFR2 signaling by inducing differential phosphorylation, and it
can be used to block angiogenesis in in vivo models of tumorigenesis.
• SRPK1 is a key regulator of the balance between two splice isoforms.
• Phosphorylation of the SRSF1 induce VEGF165 formation,
and thus lead to prostate cancer angiogenesis.

18. Detection
• In minigene assay, the amplified fragment of the analyzed gene, e.g., specific exon with
surrounding intronic sequences with and without mutations, is cloned into a special
expression plasmid enabling the analysis the pre-mRNA splicing.
• This approach can be used to confirm that the potential splicing variant affects splicing
efficiency or causes the activation of the alternative cryptic splicing sites, and to test the
role of a cis-acting elements on splicing regulation.
• The most valuable method for analysis of
splicing mutation is real-time PCR (RT-PCR).