Transcription and Post-transcription
Modification
李 希
分子医学教育部重点实验室
lixi@shmu.edu.cn
Post-transcriptional
Processing of RNA
Making ends of RNA
RNA splicing
Primary Transcript
• Primary Transcript-the initial molecule of RNA
produced--- hnRNA （heterogenous nuclear
RNA ）
• In prokaryotes, DNA →RNA →protein in
cytoplasm concurrently
• In eukaryotes nuclear RNA >> Cp RNA
Processing of eukaryotic pre-mRNA
For primary transcripts
containing multiple
exons and introns,
splicing occurs before
transcription of the gene
is complete--cotranscriptional splicing.
Human dystrophin gene has 79
exons, spans over 2,300-Kb and
requires over 16 hours to be
transcribed!
Types of RNA processing
A) Cutting and trimming to generate ends:
rRNA, tRNA and mRNA
B) Covalent modification:
Add a cap and a polyA tail to mRNA
Add a methyl group to 2’-OH of ribose in mRNA and
rRNA
Extensive changes of bases in tRNA
C) Splicing
pre-rRNA, pre-mRNA, pre-tRNA by different
mechanisms.
The RNA Pol II CTD is required for the coupling of transcription with
mRNA capping, polyadenylation and splicing
1.
The coupling allows
the processing
factors to present at
high local
concentrations
when splice sites
and poly(A) signals
are transcribed by
Pol II, enhancing the
rate and specificity
of RNA processing.
2.
The association of
splicing factors with
phosphorylated CTD
also stimulates Pol II
elongation. Thus, a
pre-mRNA is not
synthesized unless
the machinery for
processing it is
properly positioned.
Time course of RNA processing
5’ and 3’ ends of eukaryotic mRNA
5’-UTR
Add a GMP
Methylate it and
1st few nucleotides
3’-UTR
Cut the pre-mRNA
and add A’s
Capping of pre-mRNAs
• Cap=modified guanine nucleotide
• Capping= first mRNA processing event - occurs during
transcription
• CTD recruits capping enzyme as soon as it is
phosphorylated
• Pre-mRNA modified with 7-methyl-guanosine
triphosphate (cap) when RNA is only 25-30 bp long
• Cap structure is recognized by CBC（cap-binding
complex）
•
•
•
stablize the transcript
prevent degradation by exonucleases
stimulate splicing and processing
Capping of the 5’ end of nascent RNA transcripts with m7G
• The
Existing in
a single
complex
Sometimes
methylated
Sometimes
methylated
cap is added
after the nascent RNA
molecules produced
by RNA polymerase II
reach a length of 2530 nucleotides.
Guanylyltransferase is
recruited and activated
through binding to the
Ser5-phosphorylated
Pol II CTD.
• The methyl groups
are derived from Sadenosylmethionine.
• Capping helps
stabilize mRNA and
enhances translation,
splicing and export
into the cytoplasm.
Consensus sequence for 3’ process
AAUAAA: CstF (cleavage stimulation factor F)
GU-rich sequence: CPSF (cleavage and polyadenylation
specificity factor)
Polyadenylation of mRNA at the 3’ end
CPSF: cleavage and polyadenylation specificity
factor.
CStF: cleavage stimulatory factor.
CFI & CFII: cleavage factor I & II.
PAP: poly(A) polymerase.
PABPII: poly(A)-binding protein II.
RNA is cleaved 10~35-nt 3’ to A2UA3.
The binding of PAP prior to cleavage ensures
that the free 3’ end generated is rapidly
polyadenylated.
PAP adds the first 12A residues to 3’-OH
slowly.
Binding of PABPII to the initial short
poly(A) tail accelerates polyadenylation by
PAP.
Poly(A) tail stabilizes mRNA and enhances
translation and export into the cytoplasm.
The polyadenylation complex is associated with
the CTD of Pol II following initiation.
Functions of 5’ cap and 3’ polyA
• Need 5’ cap for efficient translation:
– Eukaryotic translation initiation factor 4 (eIF4)
recognizes and binds to the cap as part of initiation.
• Both cap and polyA contribute to stability of mRNA:
– Most mRNAs without a cap or polyA are degraded
rapidly.
– Shortening of the polyA tail and decapping are part of
one pathway for RNA degradation in yeast.
mRNA Half-life
• t½ seconds if seldom needed
• t½ several cell generations (i.e. ~48-72 h) for
houskeeping gene
• ≈avg 3 h in eukaryotes
• ≈avg 1.5 min in bacteria
Poly(A)+ RNA can be separated from other RNAs
by fractionation on Sepharose-oligo(dT).
Split gene and mRNA splicing
The discovery of split genes (1977)
1993 Noble Prize in Medicine
To Dr. Richard Robert and Dr. Phillip Sharp
Background: Adenovirus has a DNA genome and
makes many mRNAs. Can we determine which
part of the genome encodes for each mRNA by
making a DNA:RNA hybrid?
Experiment: Isolate Adenovirus genomic DNA,
isolate one adenovirus mRNA, hybridize and
then look by EM at where the RNA hybridizes
(binds) to the genomic DNA.
Surprise: The RNA is generated from 4
different regions of the DNA! How can we
explain this? Splicing!!
mRNA
DNA
The matured mRNAs are much shorter than
the DNA templates.
Exon and Intron
• Exon is any segment of an interrupted
gene that is represented in the mature RNA
product.
• Intron is a segment of DNA that is
transcribed, but removed from within the
transcript by splicing together the
sequences (exons) on either side of it.
Exons are
similar in size
Introns are highly
variable in size
GT-AG rule
• GT-AG rule describes the presence of these constant dinucleotides at the first
two and last two positions of introns of nuclear genes.
• Splice sites are the sequences immediately surrounding the exon-intron boundaries
• Splicing junctions are recognized only in the correct pairwise combinations
The sequence of steps in the production of mature eukaryotic mRNA as shown
for the chicken ovalbumin gene.
The consensus sequence at the exon–intron junctions of vertebrate pre-mRNAs.
4 major types of introns
4 classes of introns can be distinguished on the basis
of their mechanism of splicing and/or characterisitic
sequences:
– Group I introns in fungal mitochondria, plastids,
and in pre-rRNA in Tetrahymena (self-splicing)
– Group II introns in fungal mitochondria and
plastids (self-splicing)
– Introns in pre-mRNA (spliceosome mediated)
– Introns in pre-tRNA
Group I and II introns
The sequence of transesterification reactions that
splice together the exons of eukaryotic pre-mRNAs.
Splicing of Group I and II introns
• Introns in fungal mitochondria, plastids, Tetrahymena prerRNA
• Group I
– Self-splicing
– Initiate splicing with a G nucleotide
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis.
• Group II
– self-splicing
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism
– Does not require ATP hydrolysis
Self-splicing in pre-rRNA in Tetrahymena :
T. Cech et al. 1981
+
Exon 1
Intron 1 Exon 2
Exon 1 Exon 2
Intron 1
•Products of splicing were resolved by gel electrophoresis:
+
+
+
pre-rRNA +
Nuclear extract Additional proteins
+
+
GTP +
+
are NOT needed for
pre-rRNA
Spliced exon
Intron circle
Intron linear
splicing of this prerRNA!
Do need a G
nucleotide (GMP,
GDP, GTP or
Guanosine).
The sequence of reactions in the self-splicing of
Tetrahymena group I intron.
Where is the catalytic activity in RNase P?
RNase P is composed of a 375 nucleotide RNA and
a 20 kDa protein. The protein component will NOT
catalyze cleavage on its own.
The RNA WILL catalyze cleavage by itself !!!!
The protein component aids in the reaction but is not
required for catalysis.
Thus RNA can be an enzyme.
Enzymes composed of RNA are called ribozymes.
Hammerhead ribozymes
• A 58 nt structure is used in self-cleavage
• The sequence CUGA adjacent to stemloops is sufficient for cleavage
5'
3'
AA
A
GGCC
CCGG A
CG
U A
C G
AUC
U
G
GU A
Bond that is cle ave d.
ACCAC
C UGGUG
CUGA is r e quir e d for catalys is
Mechanism of hammerhead ribozyme
• The folded RNA forms an active site for
binding a metal hydroxide
• Attracts a proton from the 2’ OH of the
nucleotide at the cleavage site.
• This is now a nucleophile for attack on the 3’
phosphate and cleavage of the
phosphodiester bond.
1989 Nobel Prize in chemistry, Sidney Altman,
and Thomas Cech
Distribution of Group I introns
• Prokaryotes – eubacteria (tRNA & rRNA), phage
• Eukaryotes
– lower (algae, protists, & fungi)
• nuclear rRNA genes, organellar genes, Chlorella
viruses
– higher plants: organellar genes
– lower animals (Anthozoans): mitochondrial
• >1800 known, classified into ~12 subgroups, based on
secondary structure
Splicing of pre-mRNA
• The introns begin and end with almost invariant
sequences: 5’ GU…AG 3’
• Use ATP hydrolysis to assemble a large
spliceosome (45S particle, 5 snRNAs and 65
proteins, same size and complexity as ribosome)
• Mechanism is similar to that of the Group II fungal
introns:
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism for
splicing
Initiation of phosphoester transfers in pre-mRNA
• Uses 2’ OH of an A internal to the
intron
• Forms a branch point by attacking
the 5’ phosphate on the first
nucleotide of the intron
• Forms a lariat structure in the
intron
• Exons are joined and intron is
excised as a lariat
• A debranching enzyme cleaves the
lariat at the branch to generate a
linear intron
• Linear intron is degraded
Involvement of snRNAs and snRNPs
• snRNAs = small nuclear RNAs
• snRNPs = small nuclear ribonucleoproteins
particles (snRNA complex with protein)
• Addition of these antibodies to an in vitro premRNA splicing reaction blocked splicing.
• Thus the snRNPs were implicated in splicing
Role of snRNPs in RNA splicing
• Recognizing the 5’ splice site and the branch site.
• Bringing those sites together.
• Catalyzing (or helping to catalyze) the RNA cleavage.
RNA-RNA, RNA-protein and protein-protein
interactions are all important during splicing
snRNPs
U1, U2, U4/U6, and U5 snRNPs
– Have snRNA in each: U1, U2, U4/U6, U5
– Conserved from yeast to human
– Assemble into spliceosome
– Catalyze splicing
Splicing of pre-mRNA occurs in a
“spliceosome” an RNA-protein complex
spliceosome
(~100 proteins + 5 small RNAs)
pre-mRNA
spliced mRNA
The spliceosome is a large protein-RNA complex
in which splicing of pre-mRNAs occurs.
Assembly of spliceosome
• snRNPs are assembled progressively into the
spliceosome.
– U1 snRNP binds (and base pairs) to the 5’ splice site
– BBP (branch-point binding protein) binds to the branch site
– U2 snRNP binds (and base pairs) to the branch point, BBP
dissociates
– U4U5U6 snRNP binds, and U1 snRNP dissociates
– U4 snRNP dissociates
• Assembly requires ATP hydrolysis
• Assembly is aided by various auxiliary factors and
splicing factors.
Some RNA-RNA hybrids formed
during the splicing reaction
Steps of the spliceosomemediated splicing reaction
Assembly of spliceosome
A schematic diagram of six rearrangements that the spliceosome undergoes in
mediating the first transesterification reaction in pre-mRNA splicing.
The spliceosome cycle
The Significance of Gene Splicing
• The introns are rare in prokaryotic structural
genes
• The introns are uncommon in lower eukaryote
(yeast), 239 introns in ~6000 genes, only one
intron / polypeptide
• The introns are abundant in higher eukaryotes
(lacking introns are histons and interferons)
• Unexpressed sequences constitute ~80% of a
typical vertebrate structural gene
Errors produced by mistakes
in splice-site selection
Mechanisms prevent splicing error
• Co-transcriptional loading process
• SR proteins recruit spliceosome components to the 5’ and
3’ splice sites
• SR protein = Serine Arginine rich protein
• ESE = exonic splicing enhancers
• SR protein regulates alternative splicing
Alternative splicing
• Alternative splicing occurs in all metazoa and is
especially prevalent in vertebrate
Five ways to splice an RNA
Regulated alternative splicing
Different signals in
the pre-mRNA and
different proteins
cause spliceosomes
to form in particular
positions to give
alternative splicing
Alternative splicing can generate mRNAs encoding
proteins with different, even opposite functions
Fas ligand
Fas
5 6 7
(membraneassociated)
Fas pre-mRNA
5
(+)
6
7
APOPTOSIS (programmed
cell death)
(-)
Fas ligand
5 7
Soluble Fas
(membrane)
Drosophila Dscam gene contains thousands
of possible splice variants
Alternative possibilities for 4 exons leave a total number of possible
mRNA variations at 38,016. The protein variants are important for
wiring of the nervous system and for immune response.
Cis- and Trans-Splicing
Cis-: Splicing in single RNA
Trans-: Splicing in two different RNAs
Y-shaped excised introns (cis-: lariat)
Occur in C. elegance and higher eukaryotes but it does in only a
few mRNAs and at a very low level
Same splicing mechanism is
employed in trans-splicing
pre-mRNA splicing
trans-mRNA splicing
spliced leader
Spliced leader contains the cap structure!
RNA editing
• RNA editing is the process of changing the
sequence of RNA after transcription.
• In some RNAs, as much as 55% of the nucleotide
sequence is not encoded in the (primary) gene,
but is added after transcription.
• Examples: mitochondrial genes in Trypanosomes
（锥虫）
• Can add, delete or change nucleotides by editing
Two mechanisms mediate editing
• Guide RNA-directed uridine insertion
or deletion
• Site-specific deamination
Insertion and deletion of nucleotides by editing
• Uses a guide RNA
(in 20S RNP =
editosome) that is
encoded elsewhere
in the genome
• Part of the guide
RNA is
complementary to
the mRNA in vicinity
of editing
Trypanosomal RNA editing pathways.
(a) Insertion. (b) Deletion.
Mammalian example of editing
The C is converted to U in intestine by a specific
deaminating enzyme, not by a guide RNA.
Cutting and Trimming RNA
• Can use endonucleases to cut at specific sites
within a longer precursor RNA
• Can use exonucleases to trim back from the
new ends to make the mature product
• This general process is seen in prokaryotes and
eukaryotes for all types of RNA
The posttranscriptional processing of
E. coli rRNA.
RNase III cuts in stems of stem-loops
16S rRNA
23S rRNA
RNase III
No apparent primary sequence specificity - perhaps RNase III
recognizes a particular stem structure.
Eukaryotic rRNA Processing
• The primary rRNA transcript (~7500nt, 45S RNA)
contains 18S, 5.8S and 28S
• Methylation
occur mostly in rRNA sequence
80%: O2-methylribose, 20%: bases (A or G)
• peudouridine
95 U in rRNA in human are converted to Y’s
may contribute rRNA tertiary stability
Transfer RNA Processing
• Cloverleaf structure
• CCA: amino acid
binding site
• Anticodon
• ~60 tRNA genes in E.
coli
A schematic diagram of the tRNA
cloverleaf secondary structure.
Endo- and exonucleases to generate
ends of tRNA
•
•
•
Endonuclease RNase P cleaves to generate the 5’ end.
Endonuclease RNase F cleaves 3 nucleotides past the mature 3’ end.
Exonuclease RNase D trims 3’ to 5’, leaving the mature 3’ end.
Splicing of pre-tRNA
• Introns in pre-tRNA are very short (about 10-20
nucleotides)
• Have no consensus sequences
• Are removed by a series of enzymatic steps:
– Cleavage by an endonuclease
– Phosphodiesterase to open a cyclic intermediate and
provide a 3’OH
– Activation of one end by a kinase (with ATP hydrolysis)
– Ligation of the ends (with ATP hydrolysis)
– Phosphatase to remove the extra phosphate on the
2’OH (remaining after phosphodiesterase )
Steps in splicing of pre-tRNA
+
OH 5’
1. Endonuclease
Intron of
10-20
nucleotides
P
2’,3’ cyclic
phosphate
+
Excised intron
2. Phosphodiesterase
3. Kinase (ATP)
4. Ligase (ATP)
5. Phosphatase
Spliced
tRNA
CCA at 3’ end of tRNAs
• All tRNAs end in the sequence CCA.
• Amino acids are added to the CCA end during
“charging” of tRNAs for translation.
• For most eukaryotic tRNAs, the CCA is added
after transcription, in a reaction catalyzed by tRNA
nucleotidyl transferase.
All of the four bases in tRNA can be modified
Pathologies resulting from aberrant splicing
can be grouped in two major categories
• Mutations affecting proteins that are involved in splicing
Examples: Spinal Muscular Atrophy
Retinitis Pigmentosa
Myotonic Dystrophy
• Mutations affecting a specific messenger RNA and disturbing its
normal splicing pattern
Examples: β-Thalassemia
Duchenne Muscular Dystrophy
Cystic Fibrosis
Frasier Syndrome
Frontotemporal Dementia and Parkinsonism
Intron Advantage?
• One benefit of genes with introns is a phenomenon called
alternative splicing
• A pre-mRNA with multiple introns can be spliced in different
ways
– This will generate mature mRNAs with different
combinations of exons
• This variation in splicing can occur in different cell types or
during different stages of development
Intron Advantage?
• The biological advantage of alternative splicing
is that two (or more) polypeptides can be
derived from a single gene
• This allows an organism to carry fewer genes
in its genome
Do you
believe?
RNA Interference and Interference RNA
RNA interference or RNAi is a
remarkable process whereby small
noncoding RNA silence specific genes.
- RNAi was first observed in plant in immune
response to viral pathgens.
- MicroRNA regulate gene expression in
organisms from nematode to man.
Nobel Prize in Physiology or Medicine
2006, Andrew . Fire and Craig . Mello
RNA Interference: A Mechanism for
Silencing Gene Expression
1. Small dsRNA fragments can silence the expression of a
matching gene. This is RNA interference (RNAi), recently
discovered in C. elegans.
a. Injecting dsRNA into adult worms results in specific loss of the
corresponding mRNA in the worm and its progeny.
b. RNAi also occurs in many other organisms, where it protects
against viral infection and regulates developmental processes.
2. RNAi is highly specific and sensitive, with only a few
molecules of dsRNA needed, making it an excellent
research tool.
Comparison of siRNA and miRNA
siRNA
miRNA
Precursor
Endogenous or
exogenous dsRNA
Endogenous transcript
Structure
dsRNA
ssRNA
Function
mRNA cleavage
Translation inhibition and
mRNA cleavage
Target mRNA
perfect complimentarity Imperfect complimentarity
development
Inhibit transpon and
Biological
virus infection
Foreign DNA and Transgene
Foreign DNA and Transgene
Aberrant sense RNA
RdRP
dsRNA
Dicer
siRNAs
Heterochromatin formation
and Transcriptional silencing
Nature 2004,
Vol 431,
Sept.16:343
Self splicing miRNA
Mitrons :Short intronic
hairpins
RNA Ploymerase II or
III
pri-miRNA
No need of Drosha
Splicing machinery
Lariat debranching enzyme
pre-miRNAs
Cell 130, July 13
2007: 89-100
Dicer
Micro RNAs (MiRNAs)
~22NTs
RISC
lncRNA functions
Something I may not care , but you have to.