post transcriptional Modification

1. RNA SPLICING
BY
A.MANOJ KUMAR

2. ‘RNA SPLICING:-
INTRODUCTION
 RNA splicing is a form of RNA processing in
which a newly made precursor messenger RNA
(mRNA) is transformed into a mature RNA by
removing the non-coding sequences termed
introns.
 The process of RNA splicing involves the
removal of non-coding sequences or introns and
joining of the coding sequences or exons.

3.  RNA splicing takes place during or immediately after
transcription within the nucleus in the case of nucleus-
encoded genes.
 In eukaryotic cells, RNA splicing is crucial as it
ensures that an immature RNA molecule is converted
into a mature molecule that can then be translated into
proteins.
 The post-transcriptional modification is not
necessary for prokaryotic cells.
 RNA splicing is a controlled process that is regulated
by various ribonucleoproteins

4. What are Introns
 Introns are non-coding DNA sequences present
within a gene that are removed by the process of RNA
splicing during maturation of the RNA transcript.
The word ‘introns’ is used to denote both the DNA
sequences within the gene and the corresponding
sequence in RNA transcripts.
 Introns are common in the protein-coding nuclear
genes of most jawed invertebrates other eukaryotic
organisms along with unicellular organisms like
bacteria.
 Similarly, the mitochondrial genomes of jawed
vertebrates are almost entirely devoid of introns
whereas those in other eukaryotes have many introns.

5. During RNA splicing, the introns between the exons
are removed to connect two different exons that then
code for messenger RNA.
 Introns are crucial because the variation in the
protein bio-product formed is greatly enhanced by
alternative splicing in which introns take part in
prominent roles.
 Introns have a donor site (5′ end), a branch site
(near the 3′ end), and an acceptor site (3′ end) that are
required for splicing.

6. What are Exons
o Exons are protein-coding DNA sequences that contain
the necessary codons or genetic information essential
for protein synthesis.
o The word ‘exon’ represents the expressed region
present in the genome.
oThe exosome is the term used to indicate the entire
set of all exons present in the genome of the organisms.
o In genes coding for proteins, exons include both the
protein-coding sequence and the 5’ and 3’ untranslated
regions.

7. o Exons are found in all organisms ranging from jawed
vertebrates to yeasts, bacteria, and even viruses.
In the human genome, exons account for only 1% of the
total genome while the rest is occupied by intergenic
DNA and introns
o Exons are essential units in protein synthesis as they
carry regions composed of codons that code for various
proteins.
o Alternative splicing enables exons to be arranged in
different combinations, where different configuration
results in different proteins.
o A process similar to alternative splicing is exon
shuffling where exons or sister chromosomes are
exchanged during recombination.

8. SPLICEOSOME
 A spliceosome is a large and complex molecule
formed of RNAs and proteins that regulate the process
of RNA splicing.
 The spliceosome is composed of five small nuclear
RNAs (snRNA) and about 80 protein molecules.
The combination of RNAs with these proteins results in
the formation of an RNA-protein complex termed as
small nuclear ribonucleoproteins (snRNPs).
These are mostly confined within the nucleus where
they remain associated with the immature pre-RNA
transcripts.

9. These spliceosomes, in addition to working on RNA-
RNA interactions, are also involved in RNA-protein
interactions.
 The spliceosome functions as an editor that selectively
cuts out unnecessary and incorrect materials (introns) to
produce a functional final-cut.
All spliceosomes are involved in both the removal of
introns and the ligation of remaining exons.
 Another set of spliceosomes termed ‘minor
spliceosomes’ are also found in eukaryotic cells which
have less abundant RNAs and are involved in the splicing
of a rare class of pre-mRNA introns.

10. MECHANISM
1) The process of RNA splicing begins with the
binding of the ribonucleoproteins or spliceosomes
to the introns present on the splice site
2) The binding of the spliceosome results in a
biochemical process called transesterification
between RNA nucleotides.
3) During this reaction, the 3’OH group of a specific
nucleotide on the intron, which is defined during
spliceosome assembly, causes a nucleophilic attack
on the first nucleotide of the intron at the 5’ splice
site.
4) This causes the folding of the 5’ and 3’ ends,
resulting in a loop. Meanwhile, the adjacent exons
are also brought together.

11. 6) Finally, the looped intron is detached from the
sequence by the spliceosomes.
7) Now, a second transesterification reaction occurs
during the ligation of adjacent exon segments.
8) In this case, the 3’OH group of the released 5’ exon
then performs an electrophilic attack on the first
nucleotide present just behind the last nucleotide of the
intron at the 3’ splice site
9) This causes the binding of the two exon segments
along with the removal of the intron segment.

12. 10)Earlier, the intron released during splicing is
thought of as a junk unit.
11) Still, it has been recently observed that these
introns are involved in other processes related to
proteins after their removal.
12)Besides the spliceosomes, another group of
protein/ enzymes termed ‘ribozymes’ are also involved
in the control and regulation of the splicing process.

13. TYPES OF SPLICING
1.Self-splicing
 Self-splicing is a type of RNA splicing which occurs in
some rare introns that are capable of promoting
phosphodiester bond cleavage and formation without
the help of other proteins or spliceosomes.
 These introns are unique as they can mediate their
excision from precursor RNA and the subsequent
ligation of the flanking exons in a simple salt buffer.
This self-splicing reaction is facilitated by the tertiary
structure of the intron, which provides the ability to
recognize the splice sites of the precursor RNA and to
perform the cutting and ligation reactions in a very
precise manner.

14. There are three types of self-splicing introns that are
grouped as Group I, Group II, and Group III.
Group I and Group II introns perform the splicing process
in a mechanism similar to that by spliceosomes. These
suggest that these introns might be evolutionarily related to
the spliceosomes.
During self-splicing, the 5′ splice site is recognized by a
short sequence element in the intron called the internal
guide sequence.
Besides, other strongly conserved sequences of the introns
called P, Q, R, and S are needed to ‘catalyze’ the cutting and
ligation reactions.
Self-splicing follows a similar mechanism involving two
transesterification reactions resulting in the removal of
introns and ligation of exons.

15. 2.Alternative splicing- It is a splicing process resulting in
a varying composition of exons in the same RNA and
creating a range of unique proteins.
Alternative splicing of pre-mRNA is an essential
mechanism to enhance the complexity of gene
expression, and it also plays a vital role in cellular
differentiation and organism development.
Alternative splicing enables exons to be arranged in
different combinations where different configuration
results in different proteins
The process of alternative splicing might occur either by
skipping or extending some exons or by retaining
particular introns, resulting in different varieties of
mRNA formed.

16.  Regulation of alternative splicing is a complex process
in which numerous components interact with each
other, including cis-acting elements and trans-acting
factors.
 The process is further guided by the functional
coupling between transcription and splicing.
 Additional molecular features, such as chromatin
structure, RNA structure, and alternative transcription
initiation or alternative transcription termination,
collaborate with these basic components to generate
the protein diversity due to alternative splicing.

17. Alternative splicing is also essential for other functions
like the identification of novel diagnostic and prognostic
biomarkers, as well as new strategies for therapy in
cancer patients.
Thus, alternative splicing has a role in almost every
aspect of protein function, including binding between
proteins and ligands, nucleic acids or membranes,
localization, and enzymatic properties.

19. 3.tRNA splicing
• Like in mRNA, the genes in tRNA are also interrupted by
introns, but here the splicing mechanism is quite
different.
• Splicing in tRNA is catalyzed by three enzymes with
an intrinsic requirement for ATP hydrolysis.
• The process of tRNA splicing occurs in all three major
lines of descent, the Bacteria, the Archaea, and the
Eukarya, but the mechanism might differ in bacteria and
higher organisms.
• In bacteria, the introns in the tRNA are self-splicing.

20. • In Archaea and Eukarya, however, the tRNA splicing
reaction occurs in three steps where each step is
catalyzed by a distinct enzyme, each of which can
function interchangeably on all of the substrates.
 In the first step, the pre-tRNA is cleaved at the two
splice sites by an endonuclease, resulting in two tRNA
half molecules and a linear intron with 5’-OH and 3’-
cyclic PO4 ends.
 The cleavage is then followed by the ligation of the
two RNA half molecules in the presence of a tRNA ligase
enzyme.
 Finally, the PO4 ends produced from splicing are
transferred to NAD in a process catalyzed by
nicotinamide adenine dinucleotide (NAD)-dependent
phosphotransferase.

21. RNA Splicing Application
There are various biological, medical applications
associated with pre-mature RNA splicing, some of which
are:
 Pre-mRNA splicing is a fundamental process in
cellular metabolism that plays an essential role in
generating protein diversity.
 The diversity is brought about by changes in the
number and sequence of exons and introns present in
the RNA sequence.

22.  RNA splicing also helps in the regulation of gene and
protein content in the cell.
 Splicing of RNA sequences assists the process of
evolution of new and improved proteins.
Various aberrant splicing isoforms act as markers for
cancer and as targets for cancer therapy.
 Pre-mRNA splicing is a key to the pathology of
cancers where it regulates the three functional aspects
of cancer: proliferation, metastasis, and apoptosis.

23. THANK YOU…