Section O
RNA Processing and RNPs
O1 rRNA Processing
O2 tRNA Processing
O3 mRNA Processing
O4 Alternative mRNA Processing
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
O1 rRNA Processing and Ribosome
•
•
•
•
•
•
Types of RNA processing
rRNA processing in prokaryotes
rRNA processing in eukaryotes
RNPs and their study
Prokaryotic ribosomes
Euokaryotic ribosomes
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Types of RNA processing
• Definition: RNA processing is the collective term
used to describe these alterations to the primary
transcript.
• Types: The commonest types of alterations include:
(i) The removal of nucleotides by both endonucleases
and exonucleases;
(ii) The addition of nucleotides to the 5'- or 3'-ends of
the primary transcripts or their cleavage products;
(iii) The modification of certain nucleotides on either
the base or the sugar moiety.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
rRNA processing in prokaryotes
Process in E. coli :
• There are seven rRNA operons
in E. coli, each operon contains
one copy of the 5S, 16S and 23S
rRNA coding regions, together
RNase III
with some tRNA (1~4);
III P FIII
1. By RNA polymerases, an initial
30S (6000 nt) transcript is made
from one of the seven rRNA
operons;
RNase
2. This 6000 nt transcript then folds
M16 M16
and complexes with proteins;
III P F P F
M23 M23 P
3. 24 specific base methylations;
4. Cleavage by specific nucleases
(RNase III, M5, M16 and M23)
to release the mature rRNAs.
Section O: RNA Processing and RNPs.
16S RNA
23S RNA 5S RNA
Yang Xu, College of Life Sciences
rRNA processing
in eukaryotes
前18S 前5.8S 前28S
Process in mammals:
ETS2
ETS1
• rRNA genes are present in a
tandemly repeated cluster
containing 100 or more copies RNase
of the transcription unit (M2);
ITS1 ITS2
45S
1. By RNA polymerases I, an
initial 47S (13500 nt) transcript
is made from one of the units;
2. Cleavages: firstly in the
external transcribed spacers
(ETSs) 1 and 2;
47S
41S
20S
32S
3. Cleavages: then in the internal
transcribed spacer (ITSs);
4. The 5.8S region base-pair to the
28S rRNA before the mature
molecules are produced
Section O: RNA Processing and RNPs.
18S
5.8S + 28S
Yang Xu, College of Life Sciences
Prokaryotic ribosomes
The E. coli 70S ribosome is formed from a large 50S
and a small 30S sub-unit. The large sub-unit
contains 31 different proteins and one each of the
23S and 5S rRNAs. The small sub-unit contains a
16S rRNA molecule and 21 different proteins.
r RNA
Proteins
Sub-units
L1, L2, L3….L31
+
23S
5S
(2900 bases)(120 bases)
50 S
S1, S2, S3….S21
+
16S
(1540 bases)
Section O: RNA Processing and RNPs.
30S
Yang Xu, College of Life Sciences
Eukaryotic ribosomes
The complete mammalian (rat) 80S ribosome is composed of one
large 60S subunit and one small 40S subunit. The 60S subunit
contains one 23S rRNA, one 5.8S rRNA, one 5S rRNA and
about 45 proteins. The 40S subunit contains an 18S rRNA
molecule and about 30 distinct proteins.
r RNA
Proteins
Sub-units
L1, L2, L3….L45
+
23S 5.8S
5S
(4800+160bases) (120 bases)
60 S
S1, S2, S3….S30
80 S
+
18S
(1900 bases)
Section O: RNA Processing and RNPs.
40S
Yang Xu, College of Life Sciences
O2 tRNA Processing, RNase P &
Ribozymes
•
•
•
•
tRNA processing in prokaryotes
tRNA processing in eukaryotes
Rnase P
Ribozymes
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
tRNA processing in prokaryotes
The processing of tRNATyr of E. coli :
• Mature tRNAs are generated by
processing longer pre-tRNA
transcripts:
4 2
1
3’
5’
• Specific exo- and endo-nucleolytic
cleavages by RNases D, E, F and P
3
A
C
C
>150 nt
1. Endo-RNase E or F cleave 3’-end;
2. Exo-RNase D trims the 3'-end to
within 2 nt of mature length;
3. Endo-RNase P can then cut to give
the mature 5'-end;
4. Exo-RNase D finally removes the
two 3'-residues.
• Base modifications: which are
unique to each particular tRNA type.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
tRNA processing in eukaryotes
The processing of tRNATyr of yeast:
5’
• Mature tRNAs are generated by
processing longer pre-tRNA with 1. Endo-RNases
a 16 nt 5’-leader, a 14 nt intron and
a 2 nt 3’-end:
A
C
C
A
3’2. tRNA
transferase
1. Specific cleavages by endo-RNases
for 16nt 5’-leader and a 2nt 3’-end
2. tRNA transferase adds the
sequence 5'-CCA-3' to the 3'-end
to generate the mature 3'-end of
the tRNA;
3. Removal of the intron by endoRNases followed by ligation of the
half molecules of tRNA;
3. Endo-RNases
4. Base modifications: which are
unique to each particular tRNA type
(see P2).
Section O: RNA Processing and RNPs.
A A
CC
AA
U
AA
UG y
UG
G C
U A
A U
A C
U
A4. Base
U
C modifications
UAC
Yang Xu, College of Life Sciences
Ribonuclease P (RNase P)
• Definition: RNase P is an endo-nuclease composed of one RNA
molecule and one protein molecule. It is therefore a very simple RNP.
• Structure: In E. coli, RNase P is composed of a 377 nt RNA and a
small basic protein of 13.7 kDa. The secondary structure of the RNA
has been highly conserved during evolution.
• Function: Its role in cells is to generate the mature 5'-end of tRNAs
from their precursors. It is a kind of ribozymes,
• Location: RNase P are found in both prokaryotes and eukaryotes, being
located in the nucleus of the latter where they are small nuclear RNPs
(snRNPs).
• Reaction condition: The in vitro RNase P ribozyme reaction requires a
higher Mg2+ concentration than occurs in vivo, so the protein
component probably helps to catalyze the reaction in cells.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Ribozymes
Definition: Ribozymes are catalytic RNA molecules that catalyze
particular biochemical reactions.
Example: 1. RNase P is a common ribozyme that matures tRNA
that acts as an endonuclease.
2. Self-splicing intron: There is an intron in the large subunit rRNA
of Tetrahymena that can remove itself from the transcript in vitro
in the absence of protein (p534).
Function of the Protein: The in vitro reaction is about 50 times less
efficient than the in vivo reaction, so it is probable that cellular
proteins may assist the reaction in vivo.
Medical usage: Currently, there is much interest in using ribozymes
to inhibit gene expression by cleaving mRNA molecules in vivo,
as it may be possible to prevent virus replication, kill cancer cells
and discover the function of new genes by inactivating them.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
O3 mRNA Processing, hnRNPs & snRNPs
•
•
•
•
•
•
•
Processing of mRNA
hnRNP
snRNP particles
5' Capping
3' Cleavage and polyadenylation
Splicing
Pre-mRNA methylation
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Processing of mRNA of prokaryotes
• Features: There is little or no processing of mRNA transcripts
in prokaryotes. In fact, ribosomes can assemble proteins before
mRNA molecules have not yet been completely synthesized.
• Prokaryotic mRNA: It is degraded rapidly from the 5’-end and
the first cistron (protein-coding region) can therefore only be
translated for a limited amount of time.
• Stem-loop structures: Some internal cistrons are partially
protected by stem-loop structures that form at the 5'- and 3'ends and provide a temporary barrier to exonucleases and can
thus be translated more often before they are eventually
degraded.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Processing of the mRNA of Eukaryotes
• Pre-mRNAs: Those transcripts that will be processed to give
mRNAs are called pre-mRNAs.
• Processing: Pre-mRNAs are processed to mature mRNA by:
– 5’-capping;
– 3’-cleavage and polyadenylation;
– splicing and methylation.
• hnRNA:
Because eukaryotic RNA Pol II transcribes such a wide variety
of different genes, the collection of products made by RNA Pol
II is called as heterogeneous nuclear RNA (hnRNA).
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
hnRNP
hnRNP:
hnRNA + proteins  hnRNP.
hnRNA (is mainly pre-mRNA) is synthesized by RNA Pol II.
hnRNP Proteins: are classified into protein A~ protein U and
thought to help keeping the hnRNA in a single-stranded form and
to assist in the various RNA processing reactions.
The A, B and C proteins: Each of the three more abundant hnRNP
A, B and C proteins has two forms: A1, A2; B1, B2; C1, C2.
hnRNP particles: 1 RNA + 9 tetramers  1 hnRNP particle
• Purification of this material from nuclei gives a homogeneous
preparation of 30-40S particles called hnRNP particles. These
particles are about 20 nm in diameter;
• Each contains about 600-700 nt of RNA and three copies of three
different tetramers: (A1)3B2, (A2)3B2, and (C1)3C2.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
hnRNP (Function)
Slow
Fast
Hybridization of RNA molecules in vitro is stimulated by hnRNP
proteins:
• This finding suggests that hnRNP proteins prevent formation of RNA
secondary structures, thereby facilitating base pairing between
different complementary molecules.
• They may have a similar function in vivo.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
snRNP particles
snRNP: snRNA + specific proteins  snRNP.
snRNAs are mainly synthesized by RNA Pol II.
Function: The most abundant snRNPs (U1, U2, U3, U5 and U6) are
involved in splicing and methylation in pre-mRNA.
Nucleoplasmic snRNPs: The snRNPs contain the 5'-RA(U)nGR-3'
sequence and bind eight common proteins in the cytoplasm,
become hyper-methylated, and move back into the nucleus.
Construction and Work:
snRNA
snRNA
snRNP
snRNP
Section O: RNA Processing and RNPs.
splicing
Yang Xu, College of Life Sciences
5' Capping
Definition: A 7-methyl-guanosine nucleotide (m7G, or cap) is
added to the 5’-end of the transcript when it is about 25 nt long.
Feature: It is added in reverse polarity(5' to 5')
Function: It acts as:  a barrier to 5'-exonuclease attack, but it
also promotes  splicing,  transport and  translation.
A
G
3’ HO
ppp
+
p pp
T
C G
…...OH 3’
PPi
7G
mG
3’HO
Section O: RNA Processing and RNPs.
mN6A
ppp
……OH 3’
Yang Xu, College of Life Sciences
3' Cleavage and polyadenylation
• Structure of the 3’-end of mature mRNA: It is generated by
 cleavage followed by  the addition of a run of A residues
which is called the poly(A) tail.
• Function: The poly(A) tail on pre-mRNA is thought to help
stabilize the molecule since a poly(A)-binding protein can bind
to it, which should act to resist 3'-exonuclease action.
DNA
5’……AATAAA…(11-20bp)…C A……TTGTGTGTTG…3’
RNA Pol II
UG-rich
RNA
endonuclease
U7-snRNP
5’……AAUAAA…(11-20bp)…C
A……UUGUGUGUUG…3’
5’……AAUAAA…(11-20bp)…C
A……UUGUGUGUUG…3’
AAAAA(100-200A)-OH
Poly A transferase
Poly
A transferase
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Intron splicing-I
Definition: In eukaryotic pre-mRNA processing, intervening
sequences (introns) that interrupt the coding regions (exons) are
removed (spliced out), and the two flanking exons are joined.
Structure feature: This splicing reaction occurs in the nucleus and
requires the intron to have a 5‘-GU, an AG-3’, a poly-pyrimidine
tract and a branchpoint sequence (R=A,G; Y=C, U).
Intron
...CURAY …10-40 bp... (U/C)11
Exon GU
5’-end
up
branchpoint
Section O: RNA Processing and RNPs.
AG Exon
3’-end
pyrimidine down
Yang Xu, College of Life Sciences
Intron splicing-II
Function of snRNPs:
5’
The splicing reaction is catalyzed by
the Ul, U2, U4, U5, U6 snRNPs.
1. The U1 snRNP binds to the 5'splice site GU, and the U2 snRNP
binds to the branchpoint A;
5’
2. The U4, U5 and U6 can then bind,
and the intron is looped out and
the 5'- and 3'-exons close together.
3. The complex of snRNPs and premRNA is called a spliceosome.
4. After the spliceosome forms, the
two-step splicing reaction can
occur with release of the intron as
a lariat.
Section O: RNA Processing and RNPs.
GU
3’
AG
A
U1 U2
GU
3’
AG
A
U5 U4 U6
5’
AG
3’
GU
U5
A U4
U6
Spliceosome
Yang Xu, College of Life Sciences
Intron splicing-III
Process: Splicing takes place in a two-step reaction:
First, the bond in front of the G at the 5'-end of the intron at the
so-called 5'-splice site is attacked by the 2'-hydroxyl group of
the A residue of the branchpoint;
The second step, cleavage at the 3'-splice site occurs after the G
of the AG, as form and is eventually degraded.
Extron 1
Section O: RNA Processing and RNPs.
G
Extron 2
G
Extron2
外显子2
Yang Xu, College of Life Sciences
Pre-mRNA methylation
• Base methylation: Final modification or processing event that
many pre-mRNAs undergo is specific methylation of certain bases.
• mN6A: In mammalian, the most common methylation event is on
the N6 position of A residues, particularly when these A residues
occur in the sequence 5‘-RRACX-3’, where X is rarely G(R=A,G).
• Function: Up to 0.1% of pre-mRNA’s A residues are methylated,
and the methylations seem to be largely conserved in the mature
mRNA, though their function is unknown.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
O4 Alternative mRNA Processing
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Alternative splicing
• Main ways: 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 (alternative splicing). This can be
achieved in four main ways;
(i) Using different promoters
(ii) Using different poly(A) sites
(iii) Retaining certain introns
(iv) Retaining or removing certain exons.
• Mechanism: Where these events occur differently in different
cell types, it is likely that cell type-specific factors are
responsible for activating or repressing the use of processing
sites near to where they bind.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Alternative splicing
• Using different promoters: the choice of promoter (see M4) can
forces the pattern of splicing, as happens in the a-amylase and
myosin light chain genes.
• The exon is transcribed from the upstream promoter has the
stronger 5'-splice site which out-competes the downstream one
for use of the the first 3'-splice site.
Exon 1 Exon 3 Exon 4
Exon 1
P1
Exon 2
P2
Section O: RNA Processing and RNPs.
Exon 3
Exon 4
Exon 2 Exon 3 Exon 4
Yang Xu, College of Life Sciences
Alternative splicing
• Using different poly(A) sites: In the case of immunoglobulins, use
of a downstream poly(A) site includes exons encoding membraneanchoring regions, and m-Ig is produced; whereas when the
upstream site is used these regions are not present and the s-Ig is
produced.
poly(A) site
Exon 1
Exon 2
P
Section O: RNA Processing and RNPs.
Exon 3
Exon 1 Exon 2 Exon 4
Exon 4
Exon 1 Exon 2 Exon 3
Yang Xu, College of Life Sciences
Alternative splicing
• Retaining certain introns: In some situations, introns can be
retained. If the intron contains a stop codon then a truncated
protein will be produced on translation. This can give rise to an
inactive protein, as in the case of the P element transposase in
Drosophila somatic cells.
Stop codon
Exon 1 Exon 2 Exon 3
Exon 1
Exon 2
P
Section O: RNA Processing and RNPs.
Exon 3
Exon 1 Exon 2
Exon 3
Yang Xu, College of Life Sciences
Alternative splicing
• Retaining or removing certain exons: The final type of
alternative splicing illustrates that some exons can be retained
or removed in different circumstances.
• Reason: A likely reason is the existence of a factor in one cell
type that either promotes the use of a particular splice site or
prevents the use of another. The rat troponin-T pre-mRNA can
be differentially spliced in this way.
Exon 1 Exon 2 Exon 3 Exon 4
Exon 1
Exon 2
P
Section O: RNA Processing and RNPs.
Exon 3
Exon 4
Exon 1 Exon 2 Exon 4
Yang Xu, College of Life Sciences
RNA editing
Definition: This is a form of
RNA processing in which
the nucleotide sequence of
the primary transcript is
altered by either
 changing residues,
 deleting residues or
 inserting residues at
specific points along
the molecule.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
That’s all for Section O
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences