This document discusses how light regulates alternative splicing in plants through controlling transcription elongation. It presents a study that investigated the effects of light-dark conditions and histone deacetylase inhibitors on alternative splicing in Arabidopsis seedlings. The study found that light increases RNA polymerase II elongation, which regulates alternative splicing. Light-dark conditions affected alternative splicing but not total mRNA levels. The results suggest that kinetic coupling between transcription and alternative splicing is an important mechanism for plants to respond to environmental cues like light.

1. welcome

2. Presented by –
SHREYA MANDAL
Roll No: 21104 (M.Sc.)
Division of BIOCHEMISTRY
LIGHT REGULATES PLANT ALTERNATIVE
SPLICING THROUGH THE CONTROL OF
TRANSCRIPTION ELONGATION

3. INTRODUCTION
 Light induces massive reprogramming of gene
expression in plants. Light is the most crucial external
signal perceived by plants.
 Plants utilize complex photoreceptor signalling
networks to sense different light conditions and adjust
their development.
 The sensory photoreceptors and retrograde signalling
pathways co-ordinately regulate AS of pre-mRNAs.
 AS is a major post-transcriptional mechanism to
enhance the diversity of proteome in response to
environmental signals.
 AS is important for photosynthesis, flowering, defense
response and circadian clock.

4. STUDY
1
1. To investigate a putative role of chromatin structure on the
regulation of AS by light –dark conditions , Arabidopsis seedlings
are treated with histone deacetylase inhibitor trichostatin A(TSA).
2. To challenge the effect of light on AS through the control of
RNA Pol II elongation.
3. To investigate if light –dark conditions affect Pol II densities
along the genes whose AS is regulated by the same cues, ChIP

5. SPLICING
Source: http://www.plos.org

6. SPLICEOSOME MEDIATED SPLICING PROCESS:
Source: www.oxfordjournals.org

7. HISTORY OF ALTERNATIVE SPLICING
• Gilbert first discovered the alternative splicing in
bacteriophage(adenovirus) in 1977.
• The thyroid hormone calcitonin is the prominent example
of alternative splicing in mammalian cell. The gene
encodes a protein known as CGRP(calcitonin gene
related peptides).
• Drosophila melanogaster gene called Dscam, which could
potentially have 38,016 splice variants.

8. ALTERNATIVE SPLICING
Source: www.oxfordjournals.org

9. DIFFERENTIAL SPLICING

10. MECHANISM OF ALTERNATIVE
SPLICING
o The decision of splice site selection involves RNA sequence
elements and trans acting factors such as:
 Serine Arginine rich proteins
 Heterogeneous nuclear ribonucleoproteins
o Depending on the position and function of the cis -regulatory
element, they are divided into following 4 groups:
 Exonic splicing enhancers(ESEs)
 Exonic splicing silencers(ESSs)
 Intronic splicing enhancers(ISEs)
 Intronic splicing silencers(ISSs)

11. SR PROTEIN
• 200- 600 amino acids in
length.
• In 1990, SR proteins
were discovered in
Drosophila.
• The RS domain
interacts with the
hyper-phosphorylated
CTD of the largest
subunit of RNA
polymerase II during
elongation of
transcription.
Domain configuration of human serine/arginine(SR)
proteins. Serine/arginine-rich splicing factor (SRSF) 1–12
are members of the SR protein splicing family that is
defined by the N-terminal RNA recognition motifs (RRMs)
followed by a downstream arginine/serine(RS) domain.
Source:WIREs RNA 2012, 3:1–12. doi: 10.1002/wrna.10O

12. Exon-dependent splicing activation by serine/arginine (SR) proteins. Exon bound SR proteins
interact with components of the general splicing machinery via RS domain interactions. SR
protein interactions with U2 snRNP auxiliary factor (U2AF) 35 (yellow) and U1 snRNP (blue) are
indicated to facilitate 3’splice site (U2AF) or 5’splice site (U1 snRNP) recognition. The splice
junctions are indicated by AG. and GU.
WIREs : RNA 2012, 3:1–12. doi:10.1002/wrna.100

13. HETEROGENEOUS NUCLEAR
RIBONUCLEOPROTEINS(hnRNPs)
• complexes of RNA and protein
• participate during gene transcription and subsequent post-
transcriptional modification of the newly synthesized RNA (pre-
mRNA).
• responsible for suppressing RNA splicing at a particular exon.

14. Exon-dependent splicing repression by heterogeneous nuclear
ribonucleoproteins(hnRNPs) proteins. Exon bound hnRNPs proteins
interfere with the association of the general splicing machinery with the
pre-mRNA. AG and GU indicate the splice junctions.
WIREs :RNA 2012,
3:112.
doi:10.1002/wrna.100

15. REGULATION BY TRANSCRIPTION COUPLING
THE RECRUITMENT MODEL:-
 Different splicing factors
associate with the
transcription machinery
or the chromatin
template.
 Increased concentration
of these factors in the
vicinity of the pre-mRNA,
regulate splicing choices
(Das et al.,2006).
THE KINETIC MODEL:-
 The pace of transcription
elongation influences the
inclusion of alternative
exons.
 Splicing machinery is
recruited sufficiently
quickly for spliceosome
assembly and splicing to
occur ( de la Mata et al.,
2003).

16. source: Journal of
RNA POL II KINETIC MODEL FOR AS
source: Lehninger Principles of Biochemistry, Fifth Edition

17. Epigenetic modifications of histones and/or DNA via methylation lead to altered
gene expression.
Yin and chung et al., (2011) Periodontal Disease – A Clinician’s guide

18. CASE STUDY

19. MATERIALS AND METHODS
 Experimental Models:
o Arabidopsis Columbia ecotype (Col 0) was used as Wild –type.
o Mutant lines - a) hd1 mutant b) tfiis(Dolata et al.,2015)
Chemicals: a)Trichostatin A b) Camptothecin
Method : a. Radioactive RT-PCR b. q RT- PCR
c. Drug treatment d.Chromatin immuno-precipitation
e. Western blot
QUANTIFICATION AND STATISTICAL ANALYSIS :
o SMIT (Single Molecule Intron Tracking)

20. GROWTH CONDITIONS
Arabidopsis seeds were stratified for 3 days in the dark
germinated on MS medium containing 1% agar
seedlings were grown in Petri dishes (30 seeds per plate) with MS
medium at a const. temperature of 22°C under const. white light
provided by fluorescent with an intensity of irradiance between 70 and
100µmol/m² sec. for a period of 1-2 weeks.
transferred for 48 hour to darkness.
drug treatment, TSA or CPT and incubated in light or dark for additional
6 hr.
[ tfiis mutants were grown on soil for 3 weeks, incubated for 48 hr. in the
dark and then transferred to dark –light for 6 hr.]

21. RESULT - 1

22. RESULT -2

23. RESULT -3

24. CONCLUSION
 Light controls Arabidopsis nuclear AS (Petrillo et
al).Light increases Pol II elongation while in darkness
elongation is lower.
 Light regulation of AS is controlled by Pol II elongation.
 The effect of light is abolished in a mutant plant
defective in Pol II elongation.
 Changes in dark-light conditions affect AS, but not
total mRNA levels(overall transcription).

25. SUMMARY
 AS regulation by transcription elongation is a mechanism to
respond to an environmental stimulus.
 Kinetic coupling between transcription and AS is important for a
whole organism (plants) to respond to environmental cues(light).
 More efforts will be needed to understand the mechanisms
involved in light mediated RNAPII elongation.
 Nevertheless, this experimental system provides a mean to
investigate the mechanism behind the proposed regulation of AS by
transcription elongation in whole organisms..

26. Plants offer an advantage with respect with mammals to
investigate the importance of the kinetic coupling in a
whole organism in future advancements.
Future work from different groups will be needed in order
to decipher the precise nature of the mechanism
involved.