Chapter 6

RNA post-transcriptional
processing
RNA Processing
Very few RNA molecules are transcribed
directly into the final mature RNA.
Most newly transcribed RNA molecules
(primary transcripts) undergo various
alterations to yield the mature product
RNA processing is the collective term
used to describe the molecular events
allowing the primary transcripts to
become the mature RNA.
primary transcript
mature RNA.
Nucleus or Nucleolus
Cytoplasm
R
N
A

p
r
o
c
e
s
s
i
n
g

Romoval of nucleotides
addition of nucleotides
to the 5- or 3- ends
modification of certain
nucleotides
(1) Removal of nucleotides by
both endonucleases and
exonucleases
endonucleases to cut at specific
sites within a precursor RNA
exonucleases to trim the ends of a
precursor RNA
This general process is seen in
prokaryotes and eukaryotes for all
types of RNA
(2) Addition of nucleotides to
5-or 3-ends of the primary
transcripts or their cleavage
products.
Add a cap and a poly(A) tail to pre-
mRNA
(3) Modification of certain
nucleotides on either the base
or the sugar moiety.
Add a methyl group to 2-OH of ribose
in mRNA (A) and rRNA

Extensive changes of bases in tRNA
RNPs( )
Ribonucleoproteins =
RNA protein complexes
The RNA molecules in cells usually
exist complexed with proteins
specific proteins attach to specific
RNAs
Ribosomes are the largest and most
complex RNPs
3-D structure
Digital cryo-electron micrography
R
N
P

6.1: rRNA PROCESSING
rRNA processing in prokaryotes
rRNA processing in eukaryotes
6.1-1: rRNA processing in
prokaryotes
1. There are 7 different operons for rRNA
that are dispersed throughout the
genome.
2. Each operon contains one copy of each
of the 5S,the 16S and the 23S rRNA
sequences. About 1~4 coding sequences
for tRNA molecules are also present in
these rRNA operons.

3. The initial transcript has a
sedimentation coefficient of 30s (6000
nt) and is normally quite short-lived

rRNA operon
Pre-16S rRNA
Pre-tRNA Pre-23S rRNA
pre-5S rRNA
Pre-tRNA
Promoters
Terminators
RNase III, involved
in the first step of
rRNA processing

RNase M5, M16
and M23 are
involved in the
second step of
rRNA processing


30S pre-rRNA
Transcription
Pre-16S rRNA
Pre-tRNA Pre-23S rRNA
pre-5S rRNA
Pre-tRNA
Promoters
Terminators
Processing steps
Step 1: Following or during the
primary transcription, the RNA folds
up into a number of stem-loop
structures by base pairing between
complementary sequences
RNA folding
Step 2: The formation of this secondary
structure of stems and loops allows some
proteins to bind to form a RNP complex
which remain attached to the RNA and
become part of the ribosome
RNP complex
formation
Step 3: After the binding of proteins,
nucleotide modifications take place.
Example: methylation of adenine by
methylating agent S-Adenosylmethonine
(SAM)

Step 4: RNA cleavage
Pre-16S rRNA
Pre-tRNA Pre-23S rRNA pre-5S rRNA Pre-tRNA
Promoters
Terminators
30S pre-rRNA:
Transcription
Cleavage at
16S rRNA tRNA 23S rRNA 5S rRNA tRNA
RNase III III P F III III P F P E
RNase M16 M16 M23 M23 M5
rRNA operon
6.1-2: rRNA processing in
eukaryotes
rRNA in eukaryotes is also
generated from a single, long
precursor molecule by specific
modification and cleavage steps
The processes are not so well
understood
1. The rRNA genes are present in a
tandemly repeated cluster containing
100 or more copies of the
transcription unit, and are transcribed
in nucleolus by RNA Pol I
2. Precursor sizes are different among
organisms (yeast: 7000 nt;
mammalian 13500 nt), and pre-mRNA
processing is also slightly different
among organism.
3. The precursor contains
one copy of the 18S coding region and
one copy each of the 5.8S and 28S coding
regions, which together are the equivalent of the
23S rRNA in prokaryote
4. The large precursor RNA undergoes a number
of cleavages to yield mature RNA and ribosome.

5. The eukaryotic 5S rRNA
is transcribed by RNA Pol I I I from
unlinked genes to give a 121nt transcript
the transcript undergoes little or no
processing
rRNA
45SrRNA
541S rRNA
41S rRNA32S
28S5.8S rRNA20S
18S rRNA
32S28S5.8S rRNA 28S5.8S
rRNA
20S18S rRNA
18S rRNA 5.8S rRNA 28S rRNA
45S
41S
20S and 32S
Mature rRNAs
47S
18S 5.8S 28S
ETS1 ITS1 ITS2 ETS2
Indicates RNase cleavage
Mammalian pre-rRNA processing
The 5.8S region must base-pair to the 28S
rRNA before the mature molecules are
produced.
Mature rRNAs complex with protein to
form RNPs (nucleolus)
Methylation occurs at over 100 sites to
give 2-O-methylribose, which is known to
be carried out by snRNPs (nucleolus)
Introns (group I) in rRNA genes of some
lower eukarytes (Tetrahymena thermophila)
must be spliced out to generate mature
rRNAs.
Many group I introns are found to
catalyze the splicing reaction by itself in
vitro, therefore called ribozyme

6.2: tRNA PROCESSING,
RNase P AND RIBOZYMES
tRNA processing in prokaryotes
tRNA processing in eukaryotes
RNase P
Ribozymes
tRNA 3-D structure
tRNA processing in
prokaryotes
Mature tRNAs are generated by
processing longer pre-tRNA transcripts,
which involves
1. specific exo- and endonucleolytic
cleavage by RNases D, E, F and P
(general) followed by
2. base modifications which are unique to
each particular tRNA type.
Primary transcripts
RNase D,E,F and P
(See your text book)
tRNA with mature ends
Base modifications
mature tRNAs
tRNA processing in
eukaryotes
The pre-tRNA is synthesized with
a
1. 16 nt 5-leader,
2. a 14 nt intron and
3. two extra 3-nucleotides.

1. Primary transcripts forms secondary
structures recognized by endonucleases
2. 5 leader and 3 extra nucleotide removal
3. tRNA nucleptidyl transferase adds 5-
CCA-3 to the 3-end to generate the
mature 3-end
4. Intron removal

tRNA
RNAtRNA55

RNAtRNA3
RNA3
tRNA3CCA
OH


1 RNasePtRNA35
2 RNaseF3RNaseD3
RNaseD3
3 tRNA3CCA
OH
4 tRNA

1tRNA 3
2tRNA 5
RNaseIIItRNA5
RNaseP RNasePtRNA
5
RNase P
Ribonuclease P (RNase P) is an enzyme
involved in tRNA processing that removes
the 5' leader sequences from tRNA
precursors
RNase P (2)
RNase P enzymes are found in both
prokaryotes and eukaryotes, being
located in the nucleus of the latter where
they are therefore small nuclear RNPs
(snRNPs)
In E. coli, the endonuclease is composed
of a 377 nt RNA and a small basic protein
of 13.7kDa.
RNase P (1)
RNA component can catalyze pre-tRNA
in vitro in the absence of protein. Thus
RNase P RNA is a catalytic RNA, or
ribozyme.
Ribozyme (1)
Ribozymes are catalytic RNA molecules
that can catalyze particular biochemical
reactions.
RNase P RNA is a ribozyme.
RNase P RNA from bacteria is more
catalytically active in vitro than those
from eukaryotic and archaebacterial cells.
All RNase P RNAs share common
sequences and structures.
Ribozyme (2)
Self-splicing introns: the
intervening RNA that catalyze the
splicing of themselves from their
precursor RNA, and the joining of
the exon sequences
1. Group I introns, such as
Tetrahymena intron
2. Group II introns.

Ribozyme (3)
Self-cleaving RNA encoded by viral
genome to resolve the
concatameric molecules of the viral
genomic RNA
1. HDV ribozyme
2. Hairpin ribozyme
3. Hammer head ribozyme
Ribozyme (4)
Ribozymes can be used as
therapeutic agents in
1. correcting mutant mRNA in human
cells
2. inhibiting unwanted gene
expression
Kill cancer cells
Prevent virus replication
6.3: mRNA PROCESSING,
hnRNPs AND snRNPs
Processing of mRNA
hnRNP
snRNP particles
5Capping
3Cleavage and polyadenylation
Splicing
Pre-mRNA methylation
Processing of mRNA:
prokaryotes
There is essentially no processing of
prokaryotic mRNA, it can start to be
translated before it has finished
being transcribed.
Prokaryotic mRNA is degraded
rapidly from the 5 end
Processing of mRNA in
eukaryotes
In eukaryotes, mRNA is synthesized by
RNA Pol II as longer precursors (pre-
mRNA), the population of different RNA
Pol II transcripts are called
heterogeneous nuclear RNA (hnRNA).
Among hnRNA, those processed to give
mature mRNAs are called pre-mRNAs

Pre-mRNA molecules are processed
to mature mRNAs by 5-capping, 3-
cleavage and polyadenylation,
splicing and methylation.
Eukaryotic mRNA processing: overview
hnRNP: hnRNA + proteins
The hnRNA synthesized by RNA Pol II is
mainly pre-mRNA and rapidly becomes
covered by proteins to form
heterogeneous nuclear ribonucleoprotein
(hnRNP)
The hnRNP proteins are though to help
keep the hnRNA in a single-stranded
form and to assist in the various RNA
processing reactions
snRNP particles: snRNA +
proteins
1. snRNAs are rich in the base uracil,
which complex with specific proteins to
form snRNPs.
2. The most abundant snRNP are
involved in pre-mRNA splicing,
U1,U2,U4,U5 and U6.
3. A large number of snRNP define
methylation sites in pre-rRNA.
snRNAs are synthesized in the nucleus by
RNA Pol II and have a normal 5-cap.
Exported to the cytoplasm where they
associate with the common core proteins
and with other specific proteins.
Their 5-cap gains two methyl groups and
then imported back into the nucleus
where they function in splicing.
5 Capping
Very soon after RNA Pol II starts
making a transcript, and before the RNA
chain is more then 20 -30 nt long, the 5-
end is chemically modified.
7-methylguanosine is covalently to the 5
end of pre-mRNA.
Linked 5 5
Occurs shortly after initiation
7-methylguanosine (m
7
G)
Function of 5 cap
Protection from degradation
Increased translational efficiency
Transport to cytoplasm
Splicing of first exon
3 Cleavage and
polyadenylation
In most pre-mRNAs, the mature 3-end
of the molecule is generated by cleavage
followed by the addition of a run, or tail,
of A residues which is called the poly(A)
tail.
RNA polymerase II does not usually
terminate at distinct site
Pre-mRNA is cleaved ~20 nucleotides
downstream of polyadenylation signal
(AAUAAA)
~250 AMPs are then added to the 3
end
Almost all mRNAs have poly(A) tail
Function of poly(A) tail
Increased mRNA stability
Increased translational efficiency
Splicing of last intron

Splicing
the process of cutting the pre-mRNA to
remove the introns and joining together
of the exons is called splicing.
it takes place in the nucleus before the
mature mRNA can be exported to the
cytoplasm.
Introns: non-coding sequences
Exons: coding sequences
RNA splicing: removal of introns and
joining of exons
Splicing mechanism must be precise to
maintain open reading frame
Catalyzed by spliceosome (RNA
+ protein)

Biochemical steps of pre-
mRNA splicing
Step 1: a cut is made at the 5splice site,
separating the left exon and the right intron-exon
molecule. The right intron-exon molecule forms a lariat,
in which the 5terminus of the intron becomes linked by a
5-2 bond to a base within the intron. The target base is an
A in a sequence that is called the branch site
Step 2: cutting at the 3 splice site releases the free
intron in lariat form, while the right exon is
ligated (spliced) to the left exon.
C U R A Y
Lariat
Nuclear splicing occurs by two
transesterification reactions in which a free
OH end attacks a phosphodiester bond.
Spliceosome
Catalyzes pre-mRNA splicing in nucleus
Composed of five snRNPs (U1, U2, U4, U5 and
U6), other splicing factors, and the pre-mRNA
being assembled
U1 binds to the 5 splice site, then U2 to the
branchpoint, then the tri-snRNP complex of
U4, U5 and U6. As a result, the intron is looped
out and the 5- and 3 exon are brought into
close proximity.
U2 and U6 snRNA are able to catalyze the
splicing reaction.
Splicing cycle
U1 snRNA splicing
U2 snRNP
U2 snRNP
U6 snRNAU2 snRNP
U2AFU2 associated factor or
Auxiliary factor)
U4 snRNP
U4 snRNPU6 snRNPU6
snRNP U4 snRNP
U5 snRNP
U5 snRNPsnRNApre-mRNA
53
U6 snRNP
U6 snRNP5U6 snRNP
ACAGAG54749
pre-mRNA46UGU542
44 ACAUGU
GUAUGU
GAG
ACA
UAACAAAGU
U6 snRNA
5
3 5
52
48
43 38
GUAUGU
GAGACAUA
ACA
AAGU
U6 snRNA
5
3 5
52 48
43
38
U1
Cap2
U6
U2
U5
Intron
A
U6/U4 U6/U2
SnRNA U1 ~ 5, 3
consensus seq

SnRNA U2 ~
Branch Site (A
)
SnRNA U6 ~ 5
consensus seq.

SnRNA U5
5~GU & 3~AG
exons

SnRNA U6 ~ U4
SnRNA U6 ~ U2
spliceosome
Set of SR protein
(rich Ser & Arg)
5 of Intron lariat
3 of intron lariat
exons
spliceosome
lariat
Cut-ligate
Pre-mRNA methylation
The final modification or processing
event that many pre-mRNAs undergo is
specific methylation of certain bases.
The methylations seem to be largely
conserved in the mature mRNA.
6.4: ALTERNATIVE mRNA
PROCESSING
Alternative processing

Alternative poly(A) sites

Alternative splicing

RNA editing

Alternative processing
Alternative mRNA processing is the
conversion of pre-mRNA species into more
than one type of mature mRNA.
Types of alternative RNA processing
include alternative (or differential)
splicing and alternative (or
differential) poly(A) processing.

Alternative poly(A) sites
Some pre-mRNAs contain more than
one poly(A) site and these may be used
under different circumstances to
generate different mature mRNAs.
In one cell the stronger poly(A) site is
used by default, but in other cell a
factor may prevent stronger site from
being used.
Alternative splicing
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 in four ways:
(i) By using different promoters
(ii) By using different poly(A) sites
(iii) By retaining certain introns
(iv) By retaining or removing certain exons
Alternative
splicing
Alternative splicing
(A) A cassette exon can be either included
in the mRNA or excluded.
(B) Mutually exclusive exons occur when
two or more adjacent cassette exons are
spliced such that only one exon in the
group is included at a time.
(C, D) Alternative 5 and 3 splice sites
allow the lengthening or shortening of a
particular exon.
(E, F) Alternative promoters and
alternative poly(A) sites switch the 59- or
39-most exons of a transcript.
(G) A retained intron can be excised from
the pre-mRNA or can be retained in the
translated mRNA.
(H) A single pre-mRNA can exhibit
multiple sites of alternative splicing using
different patterns of inclusion.
Sex in Drosophila
is largely
determined by
alternative splicing
Sxl-Sex lethal gene: the master regulatory gene at the top of
the sex determination pathway. The downstream targets of the
Sxl protein include transcripts from the Transformer (Tra) and
Male-specific lethal 2 (Msl2) genes. Tra gene also encodes a
splicing regulator.
RNA editing
An unusual form of RNA processing
in which the sequence of the
primary transcript is altered is
called RNA editing.
Changing RNA sequence (after
transcription)
RNA editing is known to occur in two
different situations, with different
causes.
In mammalian cells there are cases in
which a substitution occurs in an individual
base in mRNA, causing a change in the
sequence of the protein that is coded.
(Base modification:A or C deamination)
In trypanosome mitochondria, more
widespread changes occur in transcripts of
several genes, when bases are
systematically added or deleted. (Base U
insertion and deletion)
1. Apolipoprotein-B mRNA in mammalian
intestine and liver. The genome contains a
single (interrupted) gene whose sequence
is identical in all tissues, with a coding
region of 4563 codons.
a protein of 512 kD representing the full coding
sequence is found in the liver.
A shorter form of the protein, ~250 kD, is
synthesized in intestine. This protein consists
of the N-terminal half of the full-length protein,
which is caused by the change of a CAA codon
to a UAA. (see Figure)
Deamination in mammalian
Liver Intestine
Deamination in mammalian
2. glutamate receptors in rat brain.
Editing at one position changes a glutamine
codon in DNA into a codon for arginine in RNA
(AI)
The change affects the conductivity of the
channel and therefore has an important effect on
controlling ion flow through the neurotransmitter.
Deamination is catalyzed by cytidine or
adenosine deaminases.
Editing enzymes are related to the general
deaminases, but have other regions or
additional subunits that control their
specificity.
Insertion or deletion of U in protozoa
Example: part of the mRNA sequence of
Trypanosome brucei coxIII shows many uridines
that are not coded in the DNA (shown in red) or that
are removed from the RNA (shown as T).
Insertion or deletion of U in protozoa
A guide RNA containing a
sequence that is complementary to
the correctly edited mRNA provides
a mechanism of U insertion or
deletion. (See figure)