This document summarizes post-transcriptional modifications in eukaryotes. It discusses how eukaryotic mRNA undergoes processing, including capping, splicing to remove introns, and polyadenylation. Splicing requires snRNPs and the spliceosome to recognize splice sites. Alternative splicing allows one gene to code for multiple proteins. tRNA and rRNA also undergo processing as they mature, including modification of bases and removal of sequences. Final mature mRNA, tRNA, and rRNA are then ready for translation.

1. POST TRANSCRIPTIONAL
MODIFICATIONS
Prepared by:
Narasimha Reddy.P.K
(2014-11-104)
college of horticulture
kerala agricultural university
Vellanikkara,thrissur
1

2. Eukaryotic vs. Prokaryotic Transcription
• In eukaryotes, transcription and translation occur in separate
compartments.
• In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is
usually monocistronic.
– Polycistronic: one mRNA codes for more than one polypeptide
– monocistronic: one mRNA codes for only one polypeptide
• “Processing” of mRNA is required in eukaryotes for the
maturation
• No processing in prokaryotes(mRNA matures on transcription)

3. Introduction…
Coupled transcription and
translation
mRNA processed and transported
out of nucleus for translation

4. 4
DNA
Introduction
Transcription
Pri -RNA
tRNA
mRNA
rRNA
Processing
Pri - transcript
Matured RNAs

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

6. 5’Capping
6
Pri-mRNA
Guanyltransferase
O-methyl transferase

7. Cap Functions
Cap provides:
1. Protection from some ribonucleases degradation
2. Stabilizes mRNA
3. Enhanced translation and splicing
4. Enhanced transport from nucleus to cytoplasm

8. mRNA splicing
• mRNA is called hnRNA (heterogenous nuclear RNA) before splicing
occurs
•The hnRNP proteins to help keep the hnRNA in a single-stranded form
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 RNA product.
• 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. 9
Splice Junction Consensus Sequence
• GU-AG rule describes the presence of these constant dinucleotides at the first
two and last two positions of introns of nuclear genes.
• Splice sites are the sequences immediately surrounding the exon-intron boundaries
• Splicing junctions are recognized only in the correct pairwise combinations

10. The sequence of steps in the production of mature eukaryotic
mRNA as shown for the chicken ovalbumin gene.

11. mRNA splicing
• Splicing is mediated by a large RNPs(Ribonucleoproteins)
complex spliceosome
• Spliceosome contains a specific set of base Uracil-rich
snRNPs (small nuclear RNPs) associated with proteins
(snRNA complex with protein)
Function of snRNPs:
• Recognizing the 5’ splice site and the branch site.
• Bringing those sites together.
• Catalyzing (or helping to catalyze) the RNA cleavage.
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12. Spliceosome Complex
• 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
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spliceosomes recognize introns starting with 5'-GU and ending in AG-3’

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

14. Mechanism of Spliceosome
U1
U4
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.
A
U5
U2
5′ 3′
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).
U6
A
U2
U6 U5
Intron plus U2,
U5, and U6
Intron will be degraded
and the snRNPs used
5′ 3′
Two connected
Exon 1 Exon 2 exons
again
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15. Alternative splicing
pre-mRNA are spliced in several different ways, allowing a single
gene to code for multiple proteins
The generation of different mature mRNAs from a particular type
of gene transcript can occur by varying the use of 5’- and 3’- splice
sites
15
Sex determination in the Drosophila

16. Polyadenylation
16
Polyadenylation Complex
Consensus sequence for 3’
process

17. Polyadenylation of mRNA at the 3’ end
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 PAP adds ≈12 A residues at a slow rate to 3’-
OH group
PABPII: poly(A)-binding protein II.
PABPII (short poly A tail) accelerates rate of addition
of A by PAP
After 200–250 A residues have been added, PABPII
signals PAP to stop polymerization
Poly (A) tail controls mRNA stability & influences
translation

18. Matured mRNA
• 5’ cap
• 5’ untranslated region
• Start codon
• Coding sequence
• Stop codon
• 3‘ untranslated region
• Poly A tail
18

19. 2. tRNA
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.
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20. Processing of tRNA
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
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21. tRNA PROCESSING AND MATURATION
Ribozyme
RNA can act as an Enzyme and catalyse reactions including its own replication

22. 3. Ribosomal RNA (rRNA)
• In cell >80% of rRNA
• Serves to release mRNA from DNA
• Act as ribozymes in protein synthesis
• Relatively G:::C rich
• Ribosome
• Prokaryotes – 70S (50S & 30S)
• Eukaryotes – 80S (60S & 40S)
• Prokaryotes – In 50S subunits - 23S & 5S :31 proteins
In 30S subunits - 16S :21 proteins
• Eukaryotes – In 60S sub-units – 28S, 5.8S and 5S :50 proteins
In 40S sub-units – 18S :33 proteins
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23. 23
Processing of ribosomal RNA
• Processing of 45s molecules occurs inside nucleolus
• 45s molecules tightly associated protein forming (RNPs)
• Frist cleavage: occurs at site I & remove 5’ terminal leader
sequence, produces 41s intermediate & 18s
• Second cleavage: occurs 41s intermediate at site 3’
generates 32s intermediate
• Final cleavage: separation of 32s intermediate into 28s, 5.8s
• Processed rRNA 28s, 5.8s & 18s

24. 2nd Cleavage
24
Processing of ribosomal RNA

25. Processing of ribosomal RNA
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26. 26
Synthesis of 5S rRNA
• rDNA cistron for 5S rRNA is present outside Nucleolar
organizer
• Transcription requires RNA pol III + TFIIIA, TFIIIB &
TFIIIC