o1 rrna processing and ribosome
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Section O RNA Processing and RNPs O1 r RNA Processing O2 t RNA Processing O3 m RNA Processing O4 Alternative mRNA Processing. O1 rRNA Processing and Ribosome. Types of RNA processing rRNA processing in prokaryotes rRNA processing in eukaryotes - PowerPoint PPT PresentationTRANSCRIPT
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
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 with some tRNA (1~4);
1. By RNA polymerases, an initial 30S (6000 nt) transcript is made from one of the seven rRNA operons;
2. This 6000 nt transcript then folds and complexes with proteins;
3. 24 specific base methylations;
4. Cleavage by specific nucleases (RNase III, M5, M16 and M23) to release the mature rRNAs.
RNase III III III IIIP F P F P F
RNaseM16 M16 M23 M23 P
23S RNA16S RNA 5S RNA
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
rRNA processing in eukaryotes
Process in mammals: • rRNA genes are present in a
tandemly repeated cluster containing 100 or more copies of the transcription unit (M2);
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 spacersexternal transcribed spacers (ETSs) 1 and 2;
3. Cleavages: then in the internal internal transcribed spacertranscribed spacer (ITSs);
4. The 5.8S region base-pair to the 28S rRNA before the mature molecules are produced
RNase
18S 5.8S + 28S
45S
41S
47SETS1 ETS2
ITS1 ITS2
前 18S 前 5.8S 前 28S
20S 32S
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
Prokaryotic ribosomesThe 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.
23S(2900 bases)
5 S(120 bases)
+
ProteinsL1, L2, L3….L31
Sub-units
50 S
16S(1540 bases)
+
S1, S2, S3….S21
r RNA
30S
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
Eukaryotic ribosomesThe 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.
5 S(120 bases)
+
ProteinsL1, L2, L3….L45
Sub-units
60 S
18S(1900 bases)
+
S1, S2, S3….S30
r RNA
40S
23S 5.8S(4800+160bases)
80 S
Section O: RNA Processing and RNPs. 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 prokaryotesThe processing of tRNATyr of E. coli :
• Mature tRNAs are generated by processing longer pre-tRNA transcripts:
• Specific exo- and endo-nucleolytic cleavages by RNases D, E, F and P
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.
ACC
>150 nt5’
3’1
3
24
• 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
5’A
3’
A AC A A U G G C U A A U A C U A U C UAC
The processing of tRNATyr of yeast:
• Mature tRNAs are generated by processing longer pre-tRNA with a 16 nt 5’-leader, a 14 nt intron and a 2 nt 3’-end:
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 endo-RNases followed by ligation of the half molecules of tRNA;
4. Base modifications: which are unique to each particular tRNA type (see P2).
1. Endo-RNases
ACC
2. tRNA transferase
3. Endo-RNases
A A C A U A G U
4. Base modifications
Section O: RNA Processing and RNPs. 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
RibozymesDefinition: 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: 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.
hnRNP
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
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.
hnRNP (Function)
Slow
Fast
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
snRNP particlessnRNP: 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: snRNAsnRNA
snRNP
snRNP
splicing
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
5' CappingDefinition: A 7-methyl-guanosine nucleotide (m7G, or cap) is added
to the 5’-end of the transcript when it is about 25 nt long.
p pp …...OH 3’
A T C G
ppp
G
3’ HO +PPi
ppp ……OH 3’
G
3’HO
m7G
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.
mN6A
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’RNA
5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’
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.
DNA 5’……AATAAA…(11-20bp)…C A……TTGTGTGTTG…3’
U7-snRNP
AAAAA(100-200A)-OHPoly A transferasePoly A transferase
• 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.
UG-richendonuclease
RNA Pol II
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).
Exon Exon
Intron
5’-endup
3’-end down
GU AG(U/C)11
pyrimidine
…10-40 bp......CURAY
branchpoint
Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences
Intron splicing-II Function of snRNPs:
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;
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 pre-mRNA is called a spliceosome.
4. After the spliceosome forms, the two-step splicing reaction can occur with release of the intron as a lariat.
5’ 3’GU AGA
U1 U2
5’ GUU5
U4U6
A
U6U4U5
5’ 3’ GU AGA
3’AG
Spliceosome
Section O: RNA Processing and RNPs. 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.
外显子 2 G G Extron2GExtron 2Extron 1
Section O: RNA Processing and RNPs. 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 -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 3 Exon 4Exon 2
P2
Exon 1
P1
Exon 1 Exon 4Exon 3
Exon 2 Exon 3 Exon 4
Section O: RNA Processing and RNPs. 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 membrane-anchoring regions, and m-Ig is produced; whereas when the upstream site is used these regions are not present and
the s-Ig is produced.
Exon 1 Exon 2P
Exon 3 Exon 4
poly(A) siteExon 1 Exon 2 Exon 4
Exon 1 Exon 2 Exon 3
Section O: RNA Processing and RNPs. 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.
Exon 2 Exon 3Exon 1
P
Stop codon
Exon 1 Exon 3Exon 2
Exon 1 Exon 2 Exon 3
Section O: RNA Processing and RNPs. 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 2Exon 1P
Exon 3 Exon 4
Exon 1 Exon 4Exon 3Exon 2
Exon 1 Exon 4Exon 2
Section O: RNA Processing and RNPs. 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
insertinginserting 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