RNA Processing

M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

Overview of the Eukaryotic mRNA
Processing
Eukaryotic cells process the RNA in the nucleus
before it is moved to the cytoplasm for protein
synthesis

The RNA that is the direct copy of the DNA is the
primary transcript

Two methods are used to process primary
transcripts to increase the stability of mRNA for its
export to the cytoplasm
RNA capping
Polyadenylation
RNA 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 the
primary transcript by the addition of a string
of As

Over all Processes
Modified guanine nucleotide
added to the 5 end
Protein-coding segment
3 UTR
Stop codon Start 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
After about 30 nt are added, 5-P is almost
immediately modified
A phosphate (terminal) is released by hydrolysis
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
Uses of Capping
Caps 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
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
50 to 250 adenine nucleotides
added to the 3 end
Protein-coding segment
Polyadenylation signal
Poly-A tail
3 UTR
Stop codon Start codon
5 Cap 5 UTR
AAUAAA AAAAAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
G P P P
5
3
Cleavage site
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/polyadenylation complex

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

(i)

(ii)
CPSF
PAP

(iii)
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
1
146
3 UTR 5 UTR
Pre-mRNA
mRNA
c) Splicing out Introns
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

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 RNA like
a cowboy rope
(U1, U2, U4, U5 and U6)
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

Thomas Cech (1981)
Nobel prize in 1989
Exception: RIBOZYME
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

Comparison
Alternative Splicing Patterns
1, 2A, 3 1, 2B, 3
1, 2A, 2B, 3 1, 3
(Calcitonin-gene related protein)
Two predominant Poly(A) sites in Rats
Cell type specific RNA splicing
Processing of pre-rRNA transcripts
Benefits of Splicing
Allows for genetic recombination
Link exons from different genes together to create a new
mRNA
Also allows for one primary transcript to encode
for multiple proteins by rearrangement of the
exons
RNA Editing
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?
Correspondence between exons
and protein domains
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 1
Domain 2
Polypeptide
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