The document discusses alternative splicing, detailing its mechanisms, regulation, and significance in gene expression, particularly in eukaryotic organisms. It explains how alternative splicing enhances protein diversity by enabling one gene to produce multiple protein isoforms through various splicing modes and regulatory factors. Additionally, the role of alternative splicing in plant adaptation to environmental stress through temperature perception is highlighted, indicating its critical function in gene regulation and proteomics expansion.

1. WELCOME

2. ALTERNATIVE SPLICING : MECHANISM AND
REGULATION
SPEAKER
SMRUTIREKHA SAHU
ROLL NO. 20950
M.Sc. 1st year
DIVISION OF BIOCHEMISTRY
CHAIRPERSON
DR. ARCHANA SACHDEV
PRINCIPAL SCIENTIST
DIVION OF BIOCHEMISTRY
CREDIT SEMINAR

3. ONE GENE MANY PROTEINS - ALTERNATIVE
SPLICING

4. EUKARYOTIC GENE STRUCTURE

5. SPLICING
• Splicing is the editing of the nascent precursor messenger RNA (pre-
mRNA) transcript into a mature messenger RNA (mRNA).
• After splicing, introns are removed and exons are joined together (ligated).
• For nuclear-encoded genes, splicing takes place within the nucleus either during
or immediately after transcription.
• For those eukaryotic genes that contain introns, splicing is usually required in
order to create an mRNA molecule that can be translated into protein.
• For many eukaryotic introns, splicing is carried out in a series of reactions
which are catalysed by the spliceosome, a complex of snRNPs. Self-splicing
introns, or ribozymes capable of catalysing their own excision from their parent
RNA molecule, also exist.

6. SPLICING

7. • The processing of bulk of eukaryotic
mRNAs is mediated by particles called
spliceosome , a large molecular complex that
recognizes sequences in the pre-mRNA,
called splice sites, to remove the noncoding
introns and join the flanking exons.
• They are ellipsoid particles of RNA and
protein (a ribonucleoprotein) and is ~25 X 50
nm in size.
• The spliceosome core consists of five small
nuclear ribonucleoproteins (snRNP1,
snRNP2, snRNP4, snRNP6, snRNP5) and
numerous spliceosome-associated factors or
proteins which assemble at introns in a
precise order.
SPLICEOSOME

8. SPLICEOSOME
MEDIATED
SPLICING
PROCESS

9. HISTORY OF ALTERNATIVE
SPLICING
• First discovered in 1977 in a bacteriophage (adenovirus) proposed by
Gilbert.
• The thyroid hormone calcitonin was found to be alternatively spliced in
mammalian cells. The primary transcript from this gene contains 6
exons; the calcitonin mRNA contains exons 1–4, and terminates after a
polyadenylation site in exon 4. Another mRNA is produced from this
pre-mRNA by skipping exon 4, and includes exons 1–3, 5, and 6. It
encodes a protein known as CGRP (calcitonin gene related peptide).
• The "record-holder" for alternative splicing is a D. melanogaster gene
called Dscam, which could potentially have 38,016 splice variants.

10. ALTERNATIVE SPLICING
• It is deviated from constitutive splicing which is a regulated process
during gene expression that results in a single gene coding for
multiple proteins wherein, particular exons maybe included or
excluded from final, processed mRNA.
• Consequently, the proteins translated from alternatively spliced
mRNAs will contain differences in their amino acid sequence and,
often, in their biological functions.
• Alternative splicing occurs as a normal phenomenon in eukaryotes,
where it greatly increases the biodiversity of proteins that can be
encoded by the genome.

11. ALTERNATIVE SPLICING

12. ALTERNATIVE SPLICING PRODUCES 3 ISOFORMS OF
PROTEIN

13. MODES OF ALTERNATIVE SPLICING
Exon skipping or cassette exon: In this case, an exon may be spliced out of the
primary transcript or retained. This is the most common mode in mammalian pre-
mRNAs
Mutually exclusive exons: One of two exons is retained in mRNAs after
splicing, but not both.

14. Alternative donor site : An alternative 5' splice junction (donor site) is used,
changing the 3' boundary of the upstream exon.
Alternative acceptor site: An alternative 3' splice junction (acceptor site) is used,
changing the 5' boundary of the downstream exon.
Intron retention: A sequence may be spliced out as an intron or simply retained.
This is distinguished from exon skipping because the retained sequence is not
flanked by introns. If the retained intron is in the coding region, the intron must
encode amino acids in frame with the neighbouring exons, or a stop codon or a
shift in the reading frame will cause the protein to be non-functional. This is the
rarest mode in mammals.

15. MECHANISM OF ALTERNATIVE
SPLICING

16. SPLICE SITE SELECTION
• The decision to include and remove exons involves RNA sequence
elements and trans acting factors such as :
 SR proteins
 hnRNPs
 RbFOX proteins
• Depending on the position and function of the cis-regulatory
elements, they are divided into 4 categories:
 ESEs(Exonic splicing enhancers)
 ESSs(Exonic splicing silencers)
 ISEs(Intronic splicing enhancers)
 ISSs(Intronic splicing silencers)

17. • They are a family of nuclear factors
functioning in both constitutive and alternative
RNA splicing.
• They bind to Exonic splicing
enhancers(ESEs) through their RNA
recognition motifs (RRMs) and mediating
protein-protein, protein-RNA interactions
through their RS (Arg-Ser repeat containing
domains).
• Structure- one or two ribonuleoprotein particle
type RNA binding domains at their amino
termini and a variable length domain enriched
in Arg-Ser dipeptides at their carboxyl termini
(RS domain) RS domains are phosphorylated
SR PROTEINS

18. U1
snRN
P
U2
snRN
P
U1
snRN
P
U1
snRN
P
U1
snRN
P
U1
snRN
P
U1
snRN
P
U1
snRN
P
U2
snRN
P
U2
snRN
P
U2
snRN
P
U2
snRN
P
U2
snRN
P
U2
snRN
P

20. • The Rbfox proteins all contain a single highly
conserved RNA recognition motif (RRM) that
specifically binds the sequences UGCAUG and GCAU
• The binding of Rbfox to a (U)GCAUG element
downstream of the alternative exon promotes its
splicing, whereas binding to an upstream element, or an
element within the exon, represses exon inclusion.
RB FOX PROTEINS

22. • Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a
family of RNA-binding proteins.
• They are multifunctional, involved not only in processing
heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but
also acting as trans-factors in regulating gene expression.
Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a
subgroup of hnRNPs, is a KH-triple repeat containing RNA-
binding protein.
• It is encoded by an intronless gene arising from hnRNP E2 through
a retrotransposition event.
• The most straightforward mode of action is based on a
competition between hnRNP proteins and other splicing factors for
binding to cis-elements having a splicing regulatory function .
Negative regulation can be achieved by occlusion of 5′ or 3′ splice
hnRNP PROTEINS

24. RNA-RNA BASE PAIRING
RNA-RNA and RNA-protein interactions that regulate mutually exclusive splicing of
Drosophila DSCAM exon 6 cluster

25. REGULATION

26. Two models have been proposed to explain the role of RNAP II in the
regulation of alternative splicing.
THE RECRUITMENT
MODEL
RNAP II and transcription
factors interact, directly or
indirectly, with splicing
factors, thereby increasing or
decreasing the efficiency of
splicing
THE KINETIC MODEL
It proposes that the rate of
transcription elongation
influences the inclusion of
alternative exons by affecting
whether the splicing machinery
is recruited sufficiently quickly
for spliceosome assembly and
splicing to occur
REGULATION BY TRANSCRIPTION
COUPLING

27. During RNA polymerase II (Pol II)-mediated transcription,
fast elongation (left) favours the recruitment of the
spliceosome to the strong 3′ splice site of a downstream
intron instead of the weak 3′ splice site of the upstream
intron, which results in exon skipping. By contrast, slow
elongation (right) favours the recruitment of spliceosome
components to the upstream intron, which results in splicing
commitment and exon inclusion
In conditions in which both 3′ splice sites are equally strong
and the upstream intron also has a binding site (green) for a
splicing factor that inhibits exon inclusion (negative factor
(NF)), fast elongation of Pol II (left) favours the recruitment of
spliceosome components to both introns, ensuring exon
definition and subsequent exon inclusion. On the contrary, slow
elongation (right) provides a time window for the negative
splicing factor to be recruited to its target site before
spliceosome components can bind to the 5′ splice site of the
downstream intron and mediate exon definition. This results in

28. Adapters that link specific histone modifications or marks to splicing factors
H3K9me2 and
H3K9me3 are
recognized and
bound by HP1α and
HP1γ, respectively.
As a result, Pol II
slows down at the
HP1-bound
chromosomal
region, leading to
increased inclusion
of a nearby
alternative exon.
Hyperacetylation
of H3 and H4
promotes a more
relaxed chromatin
structure,
increased Pol II
elongation rate
and skipping of
alternative exons
REGULATION BY CHROMATIN
STRUCTURE

29. The
chromodomain
protein MRG15
binds to
H3K36me3 and
recruits the
splicing silencer
protein PTB to
its target RNA,
thereby
promoting
skipping of an
alternative exon.
Another
H3K36me3-
binding protein,
Psip1, affects
inclusion of
alternative exons
by recruiting the
splicing regulator
SRSF1.

30. MODEL OF ALTERNATIVE SPLICING REGULATION BY DNA METHYLATION
AND CTCF ACCUMULATION DESCRIBED FOR EXON 5 (E5) OF THE CD45 GENE
E5 skipping is
favoured by fast
RNA polymerase II
(RNAPII)
elongation rates
promoted by
specific DNA
methylation in the
exonic region that
inhibits CTCF
binding. E4 and E6
skipping, on the
other hand, is
promoted by
hnRNPL binding
to pre-mRNA.
In the absence of
DNA methylation,
CTCF binds to E5
DNA where it
creates roadblocks
to RNAPII
elongation
favouring E5
recognition and
inclusion. E4 and
E6 skipping is not
affected by this
mechanism.

31. REGULATION BY ATIs AND ATTs
• These occur in the UTRs as compared to other splicing events that takes part
within the coding sequences.
• This reflects the potential regulation of large distinct groups of genes with
different mechanisms, such as strong coupling with alternative splicing in 5’
and 3’ UTRs.

32. ALTERNATIVE SPLICING AND NMD
EXON JUNCTION
COMPLEX

33. ALTERNATIVE SPLICING AND DISEASES

34. Many of these
diseases arise from
multiple distinct
molecular
mechanisms. The
exon-specific
detection of
alternative splicing
might serve as a
reliable biomarker and
provide a novel
approach to diagnose
and monitor disease
progression
ALTERNATIVE SPLICING AND DRUG
DESIGNING

35. Exitrons are internal
parts of protein-coding
exons that are hidden
in the exonic sequence
and that are
alternatively spliced.
They combine features
of both exons and
introns
ALTERNATIVE SPLICING AND DECODING GENE
EVOLUTION

36. ENVIRONMENTAL
STRESSES
Functional
full length
proteins
NMD
insensitive
mRNAs
Stable
truncate
d protein
Sequestered
intron
retaining
mRNAs
PTC+
unproductive
mRNA
Functional
fully
spliced
mRNAs
Daily
transcriptiona
l oscillations
of pre mRNA
--------------------------------------------------------------------------------
---------------------------------------------------
--------------------------
Stable oscillations
of functional mRNA
Splicing of
retained introns,
translation
^
^
Arrested splicing
storage
Possible translation of truncated proteins
Feedback to the pool of
functional mRNA
Transcript
degradation
NMD
eliciting
mRNAs
ALTERNAT
IVE
SPLICING
AND
STRESSES

37. EQUILLIBRIUM OF
FUNCTIONAL AND
PTC+ mRNAs UNDER
NORMAL CONDITIONS
INCREASE OF
FUNCTIONAL
ISOFORM
INCREASE OF PTC+
ISOFORMS
ENVIRONMEN
TAL STRESS B
ENVIRONMEN
TAL STRESS A
CONTD.

38. ABA SIGNALLING AND ALTERNATIVE
SPLICING

41. INTRODUCTION
Due to the sessile lifestyle, plants face fluctuating environmental conditions.
In order to both benefit maximally and to protect themselves from
environment, plants evolved ways to sense and responds to many
environmental cues.
Ambient temperature is one of these signals that plants sense and adapt to in
order to enhance their chance of survival and reproduction where small
changes could have major effects on plant architecture and development. One
of them being the moment of flowering.
Environmental changes trigger differential AS.
 An intron containing gene can potentially produce several to numerous
different splice forms by combining conventional splicing with alternative
selection of splice sites such as Retention of introns (RI), skipping or mutual
exclusion of exons (SE or MSE), alternative splice site selection at 5` or 3 `
end (A5 or A3).

42. STUDY
They analysed ambient temperature-directed AS in two accessions of
A. thaliana and one accession of cauliflower (B. oleracea ssp.
botrytis).
Analysis of a mutant of a splicing-related gene in A. thaliana Col-0
(SALK_144790), for which AS was observed in this accession and its
orthologous gene in cauliflower, showed an altered flowering time
response under different ambient temperatures.
This suggest that AS of splicing-related genes functions as a key
molecular mechanism in plant’s temperature response.

43. MATERIALS AND METHODS
A. thaliana Col-0 and Gy-0 seeds were sown
stratified for 2-3 days at 4ºC
transferred to 23ºC
for 3 weeks(in case of high temperature
treatment) or 6 weeks(in case of low
temperature treatment)
vegetative state -- transferred to 27˚C (Col-0
and Gy-0) or 16˚C (Col-0) for 24 hours and
then above ground parts were harvested
Brassica oleracea var. botrytis F1 seeds are
sown
transferred to the greenhouse under long day
conditions (day: 16 hours at 21˚C/night: 8
hours at 16˚C) for 2 weeks
after five weeks, half of the plants were
transferred to higher ambient temperature
(day/night: 27˚C /22˚C)
after 24 hours, meristem-enriched tissue was
harvested

44. RESULTS
The effect of ambient temperature on alternative splicing:
FIG1. A. OVERVIEW OF THE SPLICING EVENTS THAT CAN OCCUR
B. DISTRIBUTION OF DIFFERENTIAL SPLICING EVENTS UPON SHIFTS TO HIGHER OR LOWER AMBIENT

45. RESULTS
A.(TOP)RAW READS FROM
IGV
(BOTTOM)INTRON EXON
STRUCTURE WITH ALL
DETECTED SPLICING
EVENTS
B. (TOP)ALTERNATIVE
SPLICING UPON LOW
AMBIENT TEMPERATURE
IN THE IGV
(BOTTOM)THE SKIPPING
OF EXON 2 IS
DIFFERENTIAL
INDICATED BY *
C. RT-PCR
FOUR DIFFERENT
SPLICING EVENTS
RESULT IN 6 DIFFERENT
SPLICE FORMS
FIG.2

46. Splicing related genes are overexpressed:
FIG. 3 GO TERM ENRICHMENT OF DIFFERENTIALLY SPLICED
ARABISDOPSIS Col-0 GENES UPON AMBIENT TEMPEARTURE

47. The spliceosome is the target of ambient temperature-induced alternative
splicing:
FIG 4.CLASSES OF SPLICING RELATED ARABIDOPSIS Col-0 GENES THAT SHOWS DIFFERENTIAL SPLICING
OF ATLEAST ONE GENES

48. .
FIG 5. Flowering time analysis of A. thaliana Gy-0. Plants were grown
under 22°C and 27°C. Flowering time was determined by counting rosette
leaves at the moment of flower induction. N=10, mean ±SD
Alternative splicing of splicing related genes in other genetic
backgrounds:

49. A role for the differentially spliced splicing-related gene ATU2AF65A in thermosensitive
floral timing:
a. Gene structure of ATU2AF65A showing the position of TDNA insertion in SALK_144790
b. Wild type and mutant have the same phenotype at 16ºc
c. The mutant showed an increased flowering time response upon higher ambient
temperatue

50. Their study unveiled
that, upon small
temperature changes,
spliceosomal genes are
overrepresented
amongst the
differentially spliced
genes, and moreover,
that this included
many classes of
splicing related genes
thus showing that The
splicing machinery
was a target for
regulation.
The mutant for
ATU2AF65A gene
did not show any
significant differences
in expression
suggested a
biological function
for the ambient
temperature
directed splicing of
ATU2AF65A
Temperature sensing through
alternative splicing: A two-step model
CONCLUSION

51. CONCLUSION FOR THE SEMINAR
• Regulation of alternative splicing represents an important means to fine-tune gene
expression that saves time required for changes in transcriptional activation and pre-
mRNA accumulation, thus allowing rapid plant adaptation to adverse environmental
stresses.
• Individual splicing regulators control much larger group of genes than specific
transcription factors.
• The combination of alternative splicing database, tandem mass spectrometry may aid
with identification, analysis and characterization of potential alternative splicing
isoforms.
• Combining alternative splice variants dramatically expands the proteomics of
genomes.