Hello everyone, I am Dr. Ujwalkumar Trivedi, Head of Biotechnology Department at Marwadi University Rajkot. I teach Molecular Biology to the students of M.Sc. Microbiology and Biotechnology. The current presentation describes various co-transcriptional and post-transcriptional RNA modifications in eukaryotic cells. The following processes are described in detail: 1. 5' mRNA Capping 2. Splicing 3. Alternative Splicing 4. 3' Polyadenylation 5. RNA Editing Enjoy Reading.

1. Presented by
Dr. Ujwalkumar Trivedi
Assistant Professor
Department of Microbiology
Marwadi University
Rajkot (Gujarat)

3.  Primary transcript much larger than finished
product
 Precursor and partially processed RNA called
heterogeneous nuclear RNA (hnRNA)
 Processing occurs in nucleus
◦ Splicing
◦ Capping
◦ Polyadenylation

5. •Capping (addition of a
5’ 7-methyl guanosine
cap)
to remove
sequences
•Splicing
intervening
(introns)
•Polyadenylation
(addition of a poly-A
tail at the 3’)
5

6. The 5′ cap has four main
functions:
1.Regulation of nuclear export
2.Prevention of degradation
by exonucleases
3.Promotion of translation
4. Promotion of 5′ proximal
intron excision.

7.  RNA splicing is the
removal of
intervening
sequences (IVS)
that interrupt the
coding region of a
gene.
 Excision of the IVS
(intron) is
accompanied by
the precise ligation
of the coding
regions (exons).

8. •mRNA is called hnRNA (heterogenousnuclear RNA) before splicing
occurs
•The hnRNP proteins to help keep the hnRNA in a single-strandedform
and to assist in the various RNA processing reactions
• Exon and intron lengths & numbers vary in various genes
• Exon (Expressed sequences)is any segment of an interrupted gene
that is represented in the mature RNAproduct.
• Intron (intervening sequences )is a segment of DNA that is
transcribed, but removed from within the transcript by splicing
together the sequences (exons) on either side of it.
8

9.  P. Sharp and R. Roberts - 1993 Nobel Prize in Physiology &
Medicine
 Discovered using R-loop Analysis
◦ Cloned genomic DNAs of a few highly expressed nuclear
genes (e.g., hemoglobin, ovalbumin), and certain
Adenoviral genes were hybridized to RNA fractions and
visualized by EM
◦ Loops form from RNA annealing to the template strand
and displacing coding strand of DNA

10. Dotted
line is
RNA
DNA
coding
Template strand
Genomic DNA fragment containing a Globin gene was annealed to large
heterogenous nuclear RNA (hnRNA), which contained globin mRNA
precursors.
Realization of Introns

11. When genomic globin gene was annealed to cytoplasmic
mRNA (which contained mature globin mRNA) got an
internal loop of single-stranded DNA (= spliced out intron).
Fig. 14.3b
template strand
Coding strand
Realization of Introns

13. 1. Group I - common in organelles, nuclear
rRNA genes of lower eukaryotes, a few
prokaryotes
2. Group II - common in organelles, also in
some prokaryotes and archaea
3. Nuclear mRNA (NmRNA) - ubiquitous in
eucaryotes
4. Nuclear tRNA- some eukaryotes

14. 1. Each has a distinctive structure.
2. The chemistry of splicing of Groups I, II
and NmRNA is similar – i.e,
transesterification reactions
3. The splicing pathway for Group II and
nuclear mRNA introns is similar.
4. Splicing of Groups I, II and possibly
NmRNA introns are RNA-catalyzed

15. 1. Some Group I & II introns self-splice in vitro
in the absence of proteins - are
“ribozymes.
2. Conserved secondary structure but not
primary sequence.
3. Require Mg2+ to fold into a catalytically
active ribozyme.
4. Group I introns also require a guanosine
nucleotide in the first step.

16.  First self-splicing intron
discovered by T. Cech’s lab in
1981
 In the 26S rRNA gene in
Tetrahymena
 First example of a catalytic RNA!
 Nobel Prize in Chemistry to T.
Cech and S. Altman (showed that
RNase P was a true “turnover”
riboenzyme in vivo), 1989

17. GOH –
guanosine
nucleotide,
guanosine will
work because
the phosphates
don’t participate
in the reaction.
In vivo, GTP
probably used.
The 3’ terminal
G of the intron
is nearly 100%
conserved.

18. 6 domains,
“Helical Wheel”
Domain I
contains binding
sites for the 5’
exon (keeps the
5’ exon from
floating away
after the first
splicing step)
Group II intron Structure

19. 5’ ag/GUAAGU -------YNCURAC---YnNAG/g 3’
Y - pyrimidine (U or C)
Yn - string of ~ 9 pyrimidines
R - purine (A or G)
N - any base
Branch site
sequence

20. Group II Splicing
Pathway
(1) The 2’ OH of a special
internal A attacks the 5’ splice
site creating a branched intron
structure.
(2) The 3’ OH of the 5’ exon
attacks the 3’ splice site,
ligating the exons and
releasing the intron as a lariat
structure.

21. 1. Most begin with GU and end with AG.
2. Most of the internal sequences are not
conserved.
3. However, there are other important
consensus sequences near the ends (in
addition to GU and AG).

22. 5’ ag/GUAAGU -------YNCURAC---YnNAG/g 3’
Y - pyrimidine (U or C)
Yn - string of ~ 9 pyrimidines
R - purine (A or G)
N - any base
Branch site
sequence

23.  Elucidating the overall mechanism, cis
elements, and trans factors depended on:
1. Site-directed mutagenesis of genes in vitro,
and subsequent expression in vivo
(yeast, Hela cells, and others).
2. Development of accurate splicing extracts
(HeLa cells and yeast).
3. Isolation of temperature-sensitive yeast
mutants defective in NmRNA splicing.

24. Outline of the mechanisms used for three types of RNA splicing.
(A) Three types of spliceosomes. The major spliceosome (left), the AT–AC spliceosome (middle), and the trans-spliceosome
(right) are each shown at two stages of assembly. The U5 snRNP is the only component that is common to all three
spliceosomes. Introns removed by the AT–AC spliceosome have a different set of consensus nucleotide sequences from those
removed by the major spliceosome. In humans, it is estimated that 0.1% of introns are removed by the AT–AC spliceosome.
In trans-splicing, the SL snRNP is consumed in the reaction because a portion of the SL snRNA becomes the first exon of the
mature mRNA.

25. THE SPLICEOSOME
MACHINERY
RNA splicing is carried out by a large complex called
the spliceosome
snRNP (small nuclear ribonuclear proteins)

26. THE SPLICEOSOME
MACHINERY

27. 12
• Splicing snRNPs:
• U1: 5'- site recognition
• U2: branch site recognition
•U4: forms base paired
complex & acts with U6
•U5: 3'- junction binding of
U4-U6 complex
• U6: complex with U4 makes
spliceosome transesterase
spliceosomes recognize introns starting with 5'-GU and ending inAG-3’

28. U1
3′
5′
5′ splice site 3′ splice site
Branch site
A
GU
Exon 1 Exon 2
U1 binds to 5′ splicesite.
U2 binds to branch site.
AG
3′
5′
U4/U6 and U5 trimer binds. Intron loopsout
and exons are brought closer together.
U1 snRNP
A
U2 snRNP
3′
5′
A
U4/U6 snRNP
U5 snRNP
U2
Intron loops out
and exons brought
closer together
13

29. U1
U4
5′
3′
5′
5′ splice site is cut.
5′ end of intron is connected to the
A in the branch site to form a lariat.
U1 and U4 are released.
3′ splice site is cut.
Exon 1 is connected to exon 2.
The intron (in the form of a lariat) is released along with
U2, U5, and U6 (intron will be degraded).
A
U5
U6
U5
U6
U2
A
Intron plus U2,
U5, and U6
3′ Twoconnected
exons
Exon 1 Exon 2
U2
Intron will be degraded
and the snRNPs used
again
14

30.  SPLICING: The process by which introns, the noncoding
regions of genes, are excised out of the primary messenger RNA
transcript, and the exons (i.e., coding regions) are joined together
to generate mature messenger RNA. The latter serves as the
template for synthesis of a specific protein.
 ALTERNATIVE SPLICING: Alternative splicing is the
process of selecting different combinations of splice sites within
a messenger RNA precursor (pre-mRNA) to produce variably
spliced mRNAs. These multiple mRNAs can encode proteins
that vary in their sequence and activity, and yet arise from a
single gene. It was first observed in 1977.
 In 1981, the first example of alternative splicing in
a transcript from a normal, endogenous gene was characterized.

32. Humans contain approximately 250 cell types and has about 30,000 protein
coding genes responsible to encode 80,000 to 1,00,000 proteins. Alternative
splicing generates various combinations of mature mRNA thereby creating
different proteins from the same gene. It was also one the major reason to
disprove 1 gene 1 protein hypothesis.

33. 1. Exon skipping or cassette exon:
2. Mutually exclusive exons:
3. Intron retention:
4. Alternative 3’ splice site:
5. Alternative 5’ splice site:
6. Alternative polyA selection:

37. REGULATED BY: Activators and repressors.
They work in different fashion in different tissue.
They also have some protein working on it known as SR Proteins.
Their function is to ensure that alternative splicing is occurring.
They can direct the splicing machinery into different site under different
circumstances.
Thus its presence ensures a splice site would be used or not.

38.  Aberrant splicing appears to an underlying cause for an
extremely high fraction of dysfunction and disease .
 It is root in alterations of the cellular concentration,
composition, localization and activity of regulatory splicing
factors, as well as mutations in components of core splicing
machinery .
 Alternative splicing has been implicated in nearly all aspects
of cancer development, and therefore, is a main participant
in the disease.
 It is very common in cancer.

39. 16
Polyadenylation Complex
Consensus sequence for 3’
process

40. CPSF: cleavage and polyadenylation specificity factor
binds upstream AAUAAA poly(A) Signal 5’end.
CStF: cleavage stimulatory factor F interacts with a
downstream GU- sequence & bound with CPSF
forming a loop in RNA
CFI & CFII: cleavage factor I & II.
PAP: poly(A) polymerase stimulates cleavage at poly A
site
Bound PAPadds ≈12 A residues at a slow rate to3’-
OH group
PABPII: poly(A)-binding protein II.
PABPII (short poly A tail) accelerates rate of addition
of A byPAP
After 200–250 A residues have been added, PABPII
signals PAPto stop polymerization.
Poly (A) tail controls mRNA stability & influences
translation

41.  RNA editing is a molecular process through which
some cells can make discrete changes to specific
nucleotide sequences within a RNA molecule after it
has been generated by the RNA polymerase.
 It is any type of change in the RNA transcript
sequence without RNA splicing called as RNA
editing.
 RNA editing was first discovered in the mitochondria
of trypanosomes.
 RNA editing is generally done by three methods i.e.
insertion, modification or deletion of one or more
bases.

42. 1. Site specific deamination (cytosine and
adenine)
◦ Cytosine to Uracil
◦ Adenine to Inosine
2. Editing by Insertion or deletions (Guide
RNA mediated site specific insertion and
deletion of uridine bases.)

43. Apolipoprotein (apo)B
circulates in two distinct forms,
apoB100 and apoB48. Human
liver secretes apoB100, the
product of a large mRNA
encoding 4536 residues. The
small intestine of all mammals
secretes apoB48, which arises
following C-to-U deamination
of a single cytidine base in the
nuclear apoB transcript,
introducing a translational stop
codon. This process, referred to
as apoB RNA editing, operates
through a multicomponent
enzyme complex that contains
a single catalytic subunit,
apobec-1

44. Adenosine deaminases acting on RNA (ADARs)
convert adenosine to inosine in double-stranded
RNA. A-to-I editing of RNA is a widespread
posttranscriptional process that has recently
emerged as an important mechanism in cancer
biology. A-to-I editing levels are high in several
human cancers, including thyroid cancer

45. Guide RNA mediated site specific
insertion and deletion of uridine bases.
STEPS
1. Hybridisation of RNA
with gRNA.
2. Cleavage of RNA by
endonuclease.
3. Activity of TuTase and
addition of Uridine
bases.
4. Ligation of the nick
using Ligase.

46.  Essential in regulating the gene expression of an
organism.
 It is a mechanism to increase the number of different
proteins available without the need to increase the
number of the genes in the genome.
 May help to protect the genome against some viruses.

47. • 5’cap
• 5’ untranslatedregion
• Start codon
• Coding sequence
• Stop codon
• 3‘ untranslated region
• Poly Atail

48. 19
Transfer RNA/ Soluble RNA/ supernatant RNA/Adaptor
RNA
• Smallest among RNAs (75-93 nucleotides)
• Recognizes codon on mRNA
• Shows high affinity to amino acids
• Carry amino acids to the site of protein synthesis
• tRNA is transcribed by RNA polymerase III
•tRNA genes also occur in repeated copies
throughout the genome, and may contain introns.

49. 1. Removal of leader sequence &
trailer
2.Replacement of nucleotide
3.Modification of certain bases:
•Replacement of U residues at the
3′ end of pre-tRNA with a CCA
sequence.
•Addition of methyl and
isopentenyl groups to the
heterocyclic ring of purine bases.
•Methylation of the 2′-OH group
in the ribose of any residue; and
conversion of specific uridines to
dihydrouridine(D),pseudouridine(y)
4.Excision of an intron
20

50. Ribonuclease

51. adenylyl
synthetase
cyclic phosphodiesterase
intron
1
2 ligase
(2’ phosphotransferase) 3

52. Thank You
Kindly Reach us at:
Marwadi University
Rajkot-Morbi Highway Road, Gauridad, Rajkot, Gujarat 360003
Website: https://www.marwadieducation.edu.in/