RNA PROCESSING AND RNPs Section O Molecular Biology Course.
-
Upload
shawn-vincent-watkins -
Category
Documents
-
view
215 -
download
0
Transcript of RNA PROCESSING AND RNPs Section O Molecular Biology Course.
RNA PROCESSING AND RNPs
Section O
•Molecular Biology Course
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 transcriptprimary transcript
mature RNAmature RNA..
Nucleus or Nucleolus
Cytoplasm
RN
A
pro
cessin
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
endonucleasesendonucleases to cut at specific to cut at specific sites sites withinwithin a precursor RNAa precursor RNA
exonucleasesexonucleases to trim the to trim the endsends of a of a precursor RNAprecursor RNA
This general process is seen in This general process is seen in prokaryotes and eukaryotes for all prokaryotes and eukaryotes for all types of RNAtypes of RNA
(2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.
Add a cap and a Add a cap and a poly(A)poly(A) tail to pre- tail to pre-mRNAmRNA
(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
RNPsRibonucleoproteins =
RNA protein complexes
The RNA molecules in cells usually The RNA molecules in cells usually exist complexed with proteinsexist complexed with proteins
specific proteins attach to specific specific proteins attach to specific RNAsRNAs
RibosomesRibosomes are the largest and most are the largest and most complex RNPscomplex RNPs
3-D structure
Digital cryo-electron micrographyRN
P
颗粒的低温电镜
图
O1: rRNA PROCESSING AND RIBOSOMESO1: rRNA PROCESSING AND RIBOSOMES rRNA processing in prokaryotes rRNA processing in eukaryotesProkaryotic ribosomesEukaryotic ribosomes
Section O: RNA processing and RNPs
O1-1: rRNA processing in prokaryotes1. There are 7 different operons for rR
NA 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(fig1)
rRNA operonrRNA operon
Pre-16S rRNA Pre-tRNA Pre-23S rRNA pre-5S rRNA
Pre-tRNA
Promoters Terminators
O1-1: rRNA processing in prokaryotes
O1-1: rRNA processing in prokaryotes
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
O1-1: rRNA processing in prokaryotes
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
O1-1: rRNA processing in prokaryotes: 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 ERNase III III P F III III P F P E
RNase M16 M16 M23 M23 M5RNase M16 M16 M23 M23 M5
rRNA operonrRNA operon
O1-2: rRNA processing in eukaryotesrRNA 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.
O1-2: rRNA processing in Eukaryotes: rRNA features
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.
O1-2: rRNA processing in Eukaryotes: rRNA features
5. The eukaryotic 5S rRNA is transcribed by RNA Pol III from
unlinked genes to give a 121nt transcript
the transcript undergoes little or no processing
O1-2: rRNA processing in Eukaryotes: rRNA features
18S rRNA 5.8S rRNA 28S rRNA
45S
41S
20S and 32S
Mature rRNAs
47S18S 5.8S 28S
ETS1 ITS1 ITS2 ETS2
Indicates RNase cleavage
Mammalian pre-rRNA processingMammalian 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)
O1-2: rRNA processing in Eukaryotes: rRNA processing
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
O1-2: rRNA processing in Eukaryotes: rRNA processing
Protein biosynthetic machinery
Made of 2 subunits (bacterial 30S and 50S, & Eukaryotes 40S and 60S)
O1-3: Ribosomes
Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes
In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.
Prokaryotic Ribosome
E. coli ribosome is 25 nm in diameter, 2750 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+
Prokaryotic Ribosomal proteins1. Size varied from 46 aa to 557 aa.
2. Most are basic proteins which bind to RNA.
3. rRNA chaperons to assist the folding of rRNA to catalytic structure.
Ribosomal rRNAResponsible for peptide bond format
ion.
Prokaryotic Ribosome
Features of the E.coli ribosome
Cleft
PlatformCentral protuberance
Small
Stalk
Ribosome Assembly
Assembly is coupled w/ transcription and pre-rRNA processing
Prokaryotic Ribosome
Ribosome Structure (1)
Prokaryotic Ribosome
mRNA is associated with the 30S subunit
Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits.
The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.
Ribosome Structure (2)
Eukaryotic Ribosome larger and more complex than prokaryotic
ribosomes, but with similar structural and functional properties
O2: tRNA PROCESSING, RNase P AND RIBOZYMES tRNA processing in prokaryotes tRNA processing in eukaryotesRNase PRibozymes
Section O: RNA processing and RNPs
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.
tRNA processing in prokaryotes
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 processing in Eukaryotes
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 c
atalytically 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 ribozyme2. Hairpin ribozyme3. 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
O3: mRNA PROCESSING, hnRNPs AND snRNPs Processing of mRNA hnRNP snRNP particles 5’Capping 3’Cleavage and polyadenylation Splicing Pre-mRNA methylation
Section O: RNA processing and RNPs
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.
Processing of mRNA in eukaryotes
Eukaryotic mRNA processing: overview
hnRNP: hnRNA + proteins The hnRNA synthesized by RNA Pol I
I 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.
snRNP particles
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 ma
king 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
Processing of mRNA in eukaryotes
7-methylguanosine (m7G)
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.
Processing of mRNA in eukaryotes
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.
Processing of mRNA in eukaryotes
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)
Processing of mRNA in eukaryotes
Biochemical steps of pre-mRNA splicing
Processing of mRNA in eukaryotes
Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus 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.
Processing of mRNA in eukaryotes
Splicing cycle
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.
Processing of mRNA in eukaryotes
O4: 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 m
RNAs 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 exo
ns
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)