Regulation of Splicing Technique to make an RNA. Difference Between Exons and Introns. Exonic Splice Enhancers.

1. Regulation of Splicing

2. Ensuring proper splicing
exon intron T
P
E exon intron exon
P’ E
If conserved sequences dictate splice junctions, what prevents the exon 1 splice donor site from interacting with exon
3, exon 4, etc splice acceptor and branch site?

3. Exons are typically the size of 1 nucleosome
while introns are much larger
100 1000 10000
Nature 409, 860-921 (15 February 2001)
Phys Biol. 2009 Nov 24;6(4):046018.

4. Alternative Splicing can occur in multiple ways
Mol Genet Genomics. 2017 Dec;292(6):1175-1195.
Exons can be skipped or inserted
Exons can be mutually exclusive
Alternative 5’ donor sites
Alternative 5’ acceptor sites
Introns can be retained

5. Introns usually contain multiple potential
splice sites
exon intron T
exon intron exon E
Splice Donor Splice Acceptor
Splice Acceptor
Splice Acceptor
Despite the fact that introns contain multiple possible splice acceptor sites, splicing
occurs in a reproducible and conserved manner.
Other mechanisms beyond RNA sequence must define proper splicing junctions.

6. Defining exons vs. Defining introns
Intron/exon borders need to be identified.
When introns are small, the machinery seems to assemble “around” the intron
When introns are large, the machinery seems to assemble “around” the exon
Most exons are small, however when a large exon is found, it usually flanked by small introns

7. Elements within the RNA sequence can guide
splicing
• 4 Types of Splicing Regulatory Elements (SRE)
• Exonic Splicing Enhancers (ESE)
• Exonic Splicing Silencers (ESS)
• Intronic Splicing Enhancers (ISE)
• Intronic Splicing Silencers (ISS)
• These elements bind RNA binding factors
• 71 known factors that bind these elements to regulate splicing.

8. Exonic Splice Enhancers recruit SR proteins
SR proteins bind the ESE in RNA and help load the spliceosome onto the adjacent splice sites.
Recruitment is associated with activating weak splice signals.
Associated also with constitutive exons. May be a fundamental mechanism to identify exons.
SR proteins have also been associated with intronic enhancer binding.
THE ROLE OF SR AND SR-RELATED PROTEINS IN pre-mRNA SPLICING. RNA Binding Proteins, edited by Lorkovic Zdravko.
©2011 Landes Bioscience and Springer Science+Business Media.

9. SR proteins
THE ROLE OF SR AND SR-RELATED PROTEINS IN pre-mRNA SPLICING. RNA Binding Proteins, edited by Lorkovic Zdravko.
©2011 Landes Bioscience and Springer Science+Business Media.
All SR proteins have an RRM and RS
domain
RNA Recognition Motif (RRM) is responsible
for interacting with the target RNA
(exception SRSF7 which uses a zinc knuckle).
The interaction motif is degenerate allowing
SR proteins to interact with many different
sequences in virtually every exon with
varying affinities.
Arginine/Serine Domain (RS) interacts with
other RNA and protein molecules including
spliceosome components

10. SR proteins interact with the Pol II CTD and thus
participate in the coupling of transcription to
splicing

11. SR protein activity is tightly regulated
A series of phosphorylation and
dephosphorylation events regulate
both localization and activity.
Thus, the relative abundance of SR
protein activity can be adjusted
leading to strong vs weak ESE
activity.

12. Intronic Splice Enhancers associate with a
number of splicing factors
FEBS Letters Volume 581, Issue 22, 4 September 2007, Pages 4127-4131
Typically bind within the first couple hundred bases of the
intron.
Tend to promote use of the 5’ splice donor site.
Act as silencers when located closer to the 3’ exon
Thus positional location of these factors may direct activity

13. Exonic Silencing Elements interact with hnRNPs
Heterogeneous Nuclear Ribonucleoproteins have been
shown to act as splicing silencers through a variety of
mechanisms.
A. Steric repression of SR binding to a exonic splicing
enhancer
B. Promotes recruitment of other hnRNPs across the
RNA and thus silencing an enhancer
C. Interaction within an intron can block enhancer
activity or directly cover a splice donor or branch
site.
D. Formation of an RNA loop structure the blocks
recognition of an exon.

14. hnRNPs are factors important in multiple
facets of RNA regulation
• hnRNPs are protein/RNA complexes
• Aid in 3’ RNA processing
• Act as a chaperone molecule for RNA
• Promotes certain RNA structures the
either block or facilitate interactions with
other factors
• Aid in transport of RNA out of nucleus
• Regulate mRNA stability
Biochemical Journal Sep 15, 2010, 430 (3) 379-392;

15. The combination of enhancers and silencers as
well as the associating factors can direct specific
splice patterns

16. Splicing coupled to transcription is a
mechanism of regulation
First come first serve
model.
Pausing at exon borders
allows time for splice
machinery to set up.
When elongation is fast,
exons can be skipped.
Trends in Biochemical Sciences. Volume 35, Issue 9, p497–504, September 2010

17. Regulating elongation Speed: Kinetic Coupling
Nucleosome positioning:
The average exon is roughly 1 nucleosome in length
Exons on average slow transcription by 20-30 seconds
per exon.
Exons with strong splice acceptor sites have less of a
preference for a positioned nucleosome.
Genome Res. 2009 Oct; 19(10): 1732–1741.
Nature Structural & Molecular Biology 16, 996–1001 (2009)

18. Loss of histones causes exon skipping
Proc Natl Acad Sci U S A. 2015 Dec 1;112(48):14840-5.
Use of inducible H3 knockdown:
• Decreased histone occupancy at exons
• Increased rate of transcription
• Caused exon skipping

19. H3K36me3 may influence splicing
H3K36me3 is higher in exons that are more often included in the final mRNA.
H3K36me3 has a number of readers that are associated with splicing regulation
Nucleic Acids Res. 2014 Jan; 42(2): 701–713.
PTB is a protein associated with
intronic splicing silencers. It can be
recruited by MRG15, and
H3K36me3 reader
Alternatively, the H3K36me3
reader Psip1 interacts with SP
proteins to promote inclusion of
exons.

20. Histone acetylation is associated with exon
skipping
• Higher levels of histone acetylation associated with alternative exons
suggests that this mark promotes skipping.
• Histone acetylation may reduce pausing at the exon boundary

21. H3K9me3 is associated with exon retention
• H3K9me3 is normally associated with silenced heterochromatin
• Has been detected in coding regions
• Association with variable exons suggests H3K9me3 results in exon
inclusion

22. Chromatin remodeling and Splicing
• The Swi/SNF complex has been shown to interact with spliceosome
components
• Also shown to slow down rate of Pol II elongation
• Loss of Swi/SNF is associated with exon skipping
• CHD1 chromatin remodeler is associated with nucleosome
positioning.
• Loss of CHD1 resulted in decreases in H3K36me3 and intron
retention.

23. Summary of Chromatin influence on Splicing
• Through chromatin remodeling or histone modifications, chromatin
regulated splicing through:
• Altering the rate of Pol II elongation
• Recruiting splicing factors

24. The genome as a regulator of splicing
• Identification of SNPs that alter splicing
• Can impact the strength of splice donor sites,
splice acceptor sites, branch points, splicing
elements, etc …
Front. Genet., 06 July 2012

25. Elongation control does not always depend
upon amino acid abundance
Mechanisms of Microbrial Genetics: OpenStax
Riboswitches use the interaction of
the RNA with small molecules to
stabilize secondary structures.
There are a few identified
eukaryote genes that
seemingly use riboswitches to
govern splicing.