Eukaryotic pre-mRNA undergoes processing in the nucleus before being exported to the cytoplasm for protein synthesis. This involves adding a 5' cap and poly-A tail to increase stability and facilitate export. Introns are also spliced out by the spliceosome, a complex of small nuclear RNAs and proteins that cuts out introns and joins exons to form mature mRNA. Capping occurs at the 5' end shortly after transcription, while polyadenylation adds around 200 adenine nucleotides to the 3' end. Splicing removes intervening intron sequences by cutting and religating exons. These processing steps produce translation-competent mRNA from initial pre-mRNA transcripts.

1. RNA Processing

2. Overview of the Eukaryotic mRNA
Processing

3. Eukaryotic cells process the RNA in the nucleus
before it is moved to the cytoplasm for protein
synthesis
 T h e RNA that is the direct copy of the DNA ist
h
e
primary transcript
 Tw o methods are used to process primary
transcripts to increase the stability of mRNA for its
export to the cytoplasm
 RNA capping
Polyadenylation

4.  R N A capping happens at the 5’ end of the
RNA, usually adds a methylgaunosine shortly
after RNA polymerase makes the 5’ end of the
primary transcript
Splicing of introns removes the intervening
sequences in RNA
Polyadenylation modifies the 3’ end of t
h
e
primary transcript by the addition of a string
of As
Over all Processes

5. Modified guanine nucleotide
added to the 5 end
Protein-coding
segment
3 UTR
Start codon Stop codon
5 Cap 5 UTR
AAUAAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
G P P P
5
3
a) 5’ Capping of Transcript
Modified GTP
is added,
backwards, on
the 5’ end

6. After about 30 nt are added, 5’-P is almost
immediately modified
 A phosphate (terminal) is released byhydrolysis
The diphosphate 5’ end then attacks the alfa
phosphate of GTP to form a very unusual 5’-5’
triphosphate linkage – this is called condensation
This highly distinctive terminus is called a cap
The N-7 nitrogen of the terminal G is then methylated
by S-adenosyl methionine to form cap0

8. Uses of Capping
C a p s are important for subsequent
splicing reactions
They also contribute to the stability of mRNAs by
protecting their 5’ ends from phosphatases and
nucleases
In addition, caps enhance the translation of mRNA by
eukaryotic protein-synthesizing systems
Note: tRNA and rRNA molecules do not have
caps

9. b) Poly-Adenylation
Most Eukaryotic mRNAs contain poly A tail
Poly A tail is not encoded by DNA
Some mRNAs contain an internal AAUAAA (AAU
= Asn, AAA = Lys). This highly conserved
sequence is only a part of the cleavage signal,
but its context is also important
The cleavage site is 11 to 30 nt away from the
AAUAAA site on the 3’ side
After the cleavage by an endonuclease, 50 to
250 A residues are added by Poly adenylate
polymerase

10. 50 to 250 adenine nucleotides
added to the 3 end
Protein-coding segment Polyadenylation signal
Poly-A tail
3 UTR
Start codon Stop codon
5 Cap 5 UTR
AAUAAA AAA…AAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
G P P P
5
3
Cleavage site

11.  Mutating the cleavage sequence in the parent DNA will result in
mRNA that is not polyadenylated and not exported to the cytoplasm
– instead it is rapidly degraded
 A second downstream signal that is a G/U rich sequence is
required for efficient cleavage and polyadenylation, and is located
ca. 50 nucleotides from the site of cleavage.
 The cleavage and polyadenylation specficity factor (CPSF), a
large 4-subunit protein (ca. 360 kDa), forms an unstable complex
with the AAUAAA sequence that is subsequently stabilized by the
addition of at least 4 separate protein complexes that bind to the
CPSF-RNA complex.
CstF: Cleavage stimulatory factor interacts with G/U rich sequence
CFI: Cleavage factor I and CFII help stabilize protein-RNA complex
PAP: Poly(A) polymearse binds to complex before cleavage occurs
PABP: Polyadenylate-binding protein binds the Poly (A ) polymerase
Assembly of the cleavage/polyadenylatio n complex

12. Cleavage and polyadenylation Specificity Factor
Cleavage Stimualtory Factor
(PABP)
Cleavage site

13. (i)

14. (ii)
CPSF
PAP

15. (iii)

17. TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
5 Cap
Exon Intron
1
5
30 31
Exon Intron
104 105 146
Exon 3
Poly-A tail
Poly-A tail
Introns cut out and
exons spliced together
Coding
segment
5 Cap
146
3 UTR
1
5 UTR
Pre-mRNA
mRNA
c) Splicing out Introns

18. RNA splicing is responsible for the removal of the
introns to create the mRNA
Introns contain sequences that act as clues for
their removal
Carried out by assembly of small nuclear
ribonucleoprotein particles (snRNPs) –
Spliceosomes

19. Spliceosome Activity
snRNPs come together and cut out the intron
and rejoin the ends of the RNA
U1 snRNP attaches to GU of the 5’intron
U2 snRNP attaches to the branch site
U4, U5 and U6 snRNPs form a complex
bringing together both U1 and U2 snRNPs
First the donor site is cut followed by 3’ splice
site cut
Intron is removed as a lariat – loop of RNAlike
a cowboy rope

20. (U1, U2, U4, U5 and U6)

21. Mechanism of Splicing
1. The branch-point A nucleotide in the intron sequence, located close to
the 3’ splice site, attacks the 5’ splice site and cleaves it.
The cut 5’ end of the intron sequence becomes covalently linked to
this A nucleotide
2. The 3’-OH end of the first exon sequence that was created in the first
step adds to the beginning of the second exon sequence, cleaving the
RNA molecule at the 3’ splice site, and the two exons are joined

22. Self-splicing of Intron Sequences
Group I intron sequences bind a free G
nucleotide to a specific site to initiate splicing
Group II intron sequences use s specially
reactive A nucleotide in the intron sequence
itself for the same purpose
Both are normally aided by proteins that
speed up the reaction, but the reaction is
mediated by the RNA in the intron sequence
The mechanism used by Group II intron
sequences forms a lariat and resemble the
activity of spliceosomes

24. Comparison

25. Alternative Splicing Patterns

26. (Calcitonin-gene related protein)
Two predominant Poly(A) sites in Rats
Cell type specific RNA splicing

27. Processing of pre-rRNA transcripts

28. Benefits of Splicing
Allows for genetic recombination
Link exons from different genes together to create a n
e
w
mRNA
Also allows for one primary transcript to encode
for multiple proteins by rearrangement of the
exons

29. RNA Editing

30. How do mRNAs
get to the cytosol?
Why do eukaryotes have
DNA within a membrane
bound compartment and
prokaryotes do not?
Could eukaryotes function
without it?

31. Correspondence between exons
and protein domains
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide

32.  Sequences removed are called Introns
 Coding sequences flanking introns are called Exons
 Exons are not removed and are in the mRNA
 Intron removal is referred to as Splicing
 Splicing is mediated by a particle: Spliceosome
 A spliceosome is made of snRNA and protein
 There are several snRNAs in a spliceosome, U1 to U6
 Some introns have self-splicing sequences:
Ribozymes
Conclusions