[VI]. Post-Transcriptional Processing and PostTranscriptional Control of Gene Expression
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Processing of eukaryotic pre-mRNA
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About 60% of human genes give spliced mRNAs
Eukaryotic cells have evolved RNA surveillance mechanisms
that prevent incorrectly processed RNAs to be transported out
of the nucleus
Regulation of pre-mRNA processing
RNA editing
Macromolecular transport across the nuclear envelop
Cytoplasmic mechanisms of post-transcriptional regulation
Processing of rRNA and tRNA
Overview of Post-Transcriptional Control of Genes
Processing of Eukaryotic Pre-mRNA
1. Capping of mRNA
2. Splicing of mRNA
3. Polyadenylation of mRNA
Overview of mRNA Processing in Eukaryotes
Processing of pre-mRNA is co-transcriptional
Structure of the 5’
Methylated CAP
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A methyl group from Sadenosyl-methionine is added
to the N7 position of the G and
the 2’ oxygen of the 5’ ribose at
the nascent RNA
Synthesis of 5’-Cap on
Eukaryotic mRNAs
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Capping occurs shortly after initiation of
transcription
7-methyl-G is added in the 5’ end of the
nascent RNA shortly after transcription
initiates, about 25-30 nucleotides in length.
The enzyme involved in this process is a
dimeric capping enzyme associated with the
phosphorylated carboxyl-terminal domain
(CTD) of Pol II. Capping is specific for
transcripts produced by Pol II
The g-phosphate is removed from the nascent
RNA, replaced with a GMP (5’-5’ triphosphate
structure), and a methyl group from Sadenosyl-methionine is added to the N7
position of the G and the 2’ oxygen of the 5’
ribose at the nascent RNA
Capping of the nascent transcript is coupled to
elongation so that all of the transcripts will be
capped
Capping of mRNA will protect it from
degradation by 5’-exonuclease
Functions of Capping
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In prokaryotes, the ShineDalgarno sequence,
localized at 10 bases
upstream of AUG, of the
polycistronic mRNA, that
binds to 16S rRNA to initiate
translation
The AUG is localized within the consensus
sequence of GCCA/GCCAUGG (Kozak’s
sequence)
In eukaryotes, the 5’ end of the mRNA is
Capped. The CAP, after binding to the CAPbinding complex (CBC), will protect the
mRNA from been degraded by RNase
After been transported out of the nucleus,
the CBC will be replaced with eIF4E and the
complex will bind to 40S ribosome to
initiate translation
CBC contains RNA binding proteins, in
mammals encoded by CBC20/CBC80 genes
Coupling Transcription with the 5’ Capping
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Following the initiation of
transcription and of the
first few bases of the RNA,
the RNA polymerase II
pauses and only
continues transcription
once the nacent RNA has
been capped. Capping is
essential for recruitment
of the pTEF-b kinase
which is required for
transcriptional elongation
GT: Guanylyl transferase
MT: 7-methyltransferasse
RPB1 of pol II contains Tyr-ser-pro-Thr-Ser-Pro-Ser at the C-terminus
Polyadenylation
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Cleavage of RNA at the
site downstream of
AAUAAA and upstream of
a sequence rich in G/U.
These sequences are
recognized by CPSF
(cleavage- and
polyadenylation-specific
complex) and CstF
(cleavage-stimulation
factor)
Endonucleolytic cleavage
will take place
Following that,
polyadenylation will take
place in the left fragment
The right fragment will be
degraded
3’ Cleavage and Polyadenylation of PremRNA Are Tightly Coupled
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Eukaryotic mRNAs are polyadenlated except
histone mRNA
Poly(A) is added at 3’ end of the mRNA after
endonuclease cleavage of the longer RNA
transcript
An AAUAAA sequence which is 10 – 35
nucleotide upstream of the poly(A) tail is the
poly(A) signal
Second Poly(A) signal (G/U rich or U rich
sequence), about 50 nucleotides off the
cleavage site, functions for efficient cleavage
and polyadenylation
CPSF (cleavage and polyadenylation specificity
factor), a 360 kd complex consists of four
different polypeptides
CStF: cleavage stimulatory factor
CF: cleavage factor; PAP: poly(A) polymerase
PABPII: Stimulating polyadenylation
The 3′ mRNA End Processing Is Critical for
Transcriptional Termination
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2.
RNA polymerase I and III terminate their transcription upon meeting a
terminating signal on the DNA
The cleavage of pre-mRNA occurs at the site downstream of AAUAAA and
upstream of a sequence rich in G/U. These sequences are recognized by
CPSF (cleavage- and polyadenylation-specific complex) and cleavagestimulation factor (CstF)
Polyadenylation Enhances the Stability of the mRNA
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Polyadenylation of mRNA will increase the stability of mRNA from
degradation from the 3’ end
In standard histone mRNAs without polyadenylation, the mRNAs
are stablized by formation of hairpin loop during S phase
hnRNP Proteins: a diverse set of proteins with
conserved RNA binding domains associated with pre-mRNA
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Pre-mRNA during the processing are associated with many nuclear proteins,
major components of hnRNPs (Heterogenous ribonucleoprotein particles)
The nuclear RNA molecules are collectively referred as hnRNA
(heternogenous nuclear RNA) – i.e., pre-mRNA and other nuclear RNAs of
various sizes
The hnRNA with the associated proteins can be visualized by
immunostaining
Proteins of hnRNP are with sizes of 34 to 120 kD. These proteins were
isolated by irradiating the cultured cells to high-dose of UV, preparing
nuclear extracts, run the extract through an oligo-dT cellulose column,
recover the bound proteins, and then characterize the proteins.
The hnRNP proteins have a modular structure: containing one or more RNAbinding domains and at least one domain that is believed to interact with
other proteins
A diverse set of proteins with conserved RNA-binding domains associate
with pre-mRNAs
Reading List VI:
 hnRNP Complex
 Heterogenous ribonuclear particles
Functions of the hnRNP Proteins
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Interaction of the pre-mRNA with hnRNP proteins will prevent the
formation of secondary structure by pre-mRNA, thus making the
pre-mRNA accessible for interaction with other RNA molecules or
proteins
Pre-mRNA – hnRNP protein complex will make pre-mRNA a more
uniform substrate for further processing
hnRNP proteins A1, C, and D bind preferentially to the pyrimidinerich sequences at the 3’ ends of introns
The above observation suggests that different hnRNP proteins will
bind to different RNA sequences that specify RNA splicing or
cleavage/polyadenylation and contribute to the structure
recognized by RNA-processing factors.
Other studies suggest that hnRNP proteins may function in the
transport of mRNA to the cytoplasm
Conserved RNA Binding Motifs (I)
RRM Domain & Its interaction with RNA
RBD: RNA-binding
domain containing
81 amino acid
residues
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The RNA recognition motif (RRM), also called as RNP motif and the RNA
binding domain (RBP) are the most common RNA-binding domains in the
hnRNP proteins. This 80-residue domain contains two highly conserved
sequences (RNP1 and RNP2) found in yeast to human
RRM domain consists of 4-stranded β sheet flanked on one side by two α
helices. The conserved RNP1 and RNP2 sequences lie side by side on the
two central β strands and their side chains make multiple contacts with a
single-stranded region of RNA.
Conserved RNA Binding Motifs (II)
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RGG Box: This is the other RNA-binding motif found in
hnRNP proteins, containing five Arg-Gly-Gly (RGG)
repeats with several interspersed aromatic amino acids
 This motif is similar to the RNA-binding domains of the HIV
Tat protein
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The KH motif: a 45-residue motif is found in the
hnRNP K protein and several other RNA-binding
proteins, commonly two or more copies of the KH
motif are interspersed with RGG repeats
 The 3D-structure of KH domain is similar to that of the RRM
domain but smaller. It consists of three b-sheet structure
supported from one side by 2 a-helices
 RNA binds to the KH motif by interacting with a hydrophobic
surface formed by the α helices and one β-strand
Splicing: RNA-DNA Hybridization to Introns Are
Spliced from Pre-mRNA
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(a) Eco RI fragment of
adenovirus DNA containing
exon gene. The gene
contains 4 short exons and
three introns
(b) Electron micrograph (left)
and schematic drawing
(right) of hybrid between
DNA and RNA
Introns in the pre-mRNA are
removed by RNA splicing
Richard Roberts and Philip
Sharp were awarded with a
Nobel prize in 1993 for the
discovery of splicing of
precursor mRNA
For long transcription units, splicing of introns in the nascent RNA begins
before the entire transcription is completed
Reading List VI: (i). R Loop Mapping; (ii) Nobel Lecture by Richard Sharp; (iii)
Mapping of viral RNA with viral DNA
Consensus Sequences around 5’ and 3’ Splice
Sites in Vertebrate Pre-mRNAs
Donor
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Acceptor
Location of specific splice sites can be determined by comparing the
genomic and specific cDNA sequences
A pyrimidine rich region (~15 bases) is located in the upstream of the
3’ splice site
About 30 – 40 nucleotides at each end of an intron are necessary for
splicing to occur at normal rates
Donor splice site: GU; Acceptor splice site: AG; This is termed GU/AG
rule
Assigned Reading:
 Intron splicing
Two Transesterification Reactions in
Splicing of Exons
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Two sequential transesterification
reactions are required to remove
the intron sequence
Introns are removed as a lariat-like
structure in which the 5’ G of the
intron is joined in an unusual 2’,5’phosphoester bond to an
adenosine near the 3’ end of the
intron. This A is called the branch
point.
Since the number of the
phosphodiester bond in the RNA
molecule during splicing is not
changed, the process does not
require energy
Two Steps of Transesterification Reactions
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The consensus sequence of the branch
site in yeast is UACUAAC, and in
multicellular eukaryotes is purine and
pyrimidines at each position
First step of splicing reaction is
nucleophilic attack by the 2’-OH
on the 5’ splice site
The left exon takes the formof a
linear molecule
The right intron exon molecule
forms a lariat, in which the
5’terminus generated at the end of
the intron simultaneously
transesterificates to become
linked by a 2’-5’ bond to a base
within the intron. The target base
“A” in a sequence that is called
the “branch site”
In the second step, the free 3’-OH
of the exon that was released by
the first reaction attacks the bond
at the 3’ splice site to form a 3’-5’
phosphoester bond
Structure of snRNA Molecule
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snRNA
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U1 snRNP contains the core
Sm proteins, three U1-specific
proteins (U1 – 70K, U1A and
U1C) and U1 snRNA
U1 snRNA contains several
domains:
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Sm-binding site: interacting
with the common snRNP
proteins
 Stem-loop structures: binding
to proteins that are unique to
U1 snRNP
U1 snRNA interacts with the 5’
splice site by base pairing between
its single-stranded 5’-terminus and
a stretch of four to six bases of the
5’splice site
Mutations in the 5’splice site and
U1 snRNA can be used to test the
importance of pairing of 5’ splice site and the snRNA
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snRNAs Base-Pairing with Pre-mRNA
During Splicing
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Splicing requires the presence of small nuclear RNA (snRNA)
Five U-rich small nuclear RNAs (snRNA), designated as U1, U2, U4,
U5 and U6, participate in pre-mRNA splicing
These RNAs are 107 to 210 nucleotides long, associated with 6 to 10
proteins in small nuclear ribonucleoprotein particles (snRNPs)
Spliceosome
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Small cytoplasmic RNAs (scRNA;
scyrps) – RNAs that are present in the
cytoplasm (and sometimes are also
found in the nucleus).
Small nuclear RNA (snRNA; snurps) –
One of many small RNA species
confined to the nucleus; several of
them are involved in splicing or other
RNA processing reactions.
Small nucleolar RNA (snoRNA) – A
small nuclear RNA that is localized in
the nucleolus.
The five snRNPs involved in splicing are U1, U2, U5, U4, and U6.
Together with some additional proteins (splicing factors), the
snRNPs form the spliceosome (~12 MDa).
Figure in the left shows the number of proteins present in the
sliceosome
Formation of the Commitment Complex
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SR proteins: Proteins that play a critical role in the formation of
spliciosomes. These proteins are splicing factors that contains one
or two RNA recognition domains (RS domains; Arg/Ser repeats)
U1 snRNP initiates splicing by binding to the 5′ splice site by
means of an RNA–RNA pairing reaction.
The commitment complex (or E complex) contains U1 snRNP
bound at the 5′ splice site and the protein U2AF bound to a
pyrimidine tract between the branch site and the 3′ splice site.
Intron Definition/Exon Definition (I)
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In multicellular
eukaryotic cells
where splicing
signals are
highly variable,
SR proteins play
an essential role
in initiating the
formation of the
splicing
commitment
complex
In yeast, all intron containing genes are interrupted by a single small
intron, the 5’ and 3’ splice sites are recognized by U1 snRNP, BBP and
Mud2. this is referred as “intron definition”
The figure in the right shows exon definition which happens in short
exons and long introns. More details are explained in the next slide
Intron Definition/Exon Definition (II)
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Intron definition mechanism also applies to splicing of small
introns in multicellular eukaryotic cells
Many muticellular eukaryotic genes possess long introns with
many sequences resemble the real splice sites. To ensure correct
recognition of the splice sites, the mechanism of “exon definition”
is employed
In exon definition, the U2AF heterodimer (U2 snRNP auxiliary
factor) binds to the 3’splice site and U1 snRNP base pairs with the
5’ splice site downstream from the exon sequence. This process
may be aided by SR proteins that bind to specific exon sequences
between the 3’ and downstream 5’ specific sites. By an unknown
mechanism, the splicing is done properly
The Spliceosome
Assembly Pathway
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from U6 snRNP to allow U6 snRNA
to pair with U2 snRNA to form the
catalytic center for splicing.
Both transesterification reactions
take place in the activated
spliceosome (the C complex).
The splicing reaction is reversible at
all steps.
E complex progresses to prespliceosome (the A complex) in
the presence of ATP.
Recruitment of U5 and U4/U6
snRNPs converts the prespliceosome to the mature
spliceosome (the B1 complex).
The B1 complex is next
converted to the B2 complex in
which U1 snRNP is released to
allow U6 snRNA to interact with
the 5′ splice site.
The final step of transesterification is the formation of the lariate
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The commitment U4 dissociates
Splicing Utilizes a Series of Base Pairing
Reactions between snRNAs and Splice Sites
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U1 pairs with 5’ splice
site
U2 pairs with the branch
site
U6 pairs with 5’ splice
site
U5 is close to both exons
Mutation that Affects the Binding of SR Proteins to the
Exonic Splicing Enhancer
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Mutation in SR protein that results in unable to bind to the exonic
splicing enhancer-------resulting skipping exons during splicing. The
truncated mRNA will be degraded or translated into protein with
abnormal function
For example, spinal muscle atrophy, a disease that causes childhood
mortality, is resulted from mutation of SR protein that fails to bind to the
exonic splicing enhancer of SMN2 pre-mRNA and causes exon skipping
leading to low production of SMN2 protein.
In childhood, low levels of SMN2 protein will lead to low viability of
spinal cord motor neurons, resulting in death.
Approximately 15% of the single
base-pair mutations that cause
human genetic diseases interfere
with proper exon definition.
Although some of the mutation may
lead to use of alternative splice sites,
others will result in skipping exons
due to SR protein failing to bind to
the exonic splicing enhancer (a sixbase motif)
Splicing Can Also Occur Between AU and AC
A minority of introns begin with AU and AC as opposed to the GU and
AG found in most introns. Removal of these types of introns involves
lariat formation and the U5 RNA, but the other U RNAs involved are
different in the two cases. The AU-AC type splicing takes placing in
the cytoplasm, suggesting that this type of splicing may have
different function than the GU-AG type of splicing in the nucleus.
Discovery of a Hammerhead Ribozyme
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Thomas Cech discovered an RNA molecule that possessed enzymatic
activity that could remove intervening sequence in tetrahymena rRNA–
this discovery led to a Nobel prize in 1989
Self Splicing Introns (Ribozymes) : Introns that can be spliced out in the
absence of splicing protein factors in vitro
Hammerhead Ribozyme
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Reading List VI:
(i) Cech’s Nobel Prize lecture
(ii) Hammerhead ribozyme
Self-Splicing of Group II and Group I Introns
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Group I introns (present in nuclear rRNA
genes of protozoans) and group II
introns (present in protein-coding genes
and some rRNA and tRNA genes in
mitochoria and chloroplasts of plants
and fungi)
Group II introns excise themselves from
RNA by an autocatalytic splicing event
(autosplicing or self-splicing).
The splice junctions and mechanism of
splicing of group II introns are similar to
splicing of nuclear introns.
A group II intron folds into a secondary
structure that generates a catalytic site
resembling the structure of U6-U2nuclear intron.
Group I introns excise themselves from
RNA also by a autocatalytic splincing
event
Comparison of Self-Splicing Group II Introns
and U snRNAs in Spliceosome
First Transesterification
Second
Transesterification
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Two types of self splicing introns have been discovered: Group I
introns (present in nuclear rRNA genes of protozoans) and group
II introns (present in protein-coding genes and some rRNA and
tRNA genes in mitochoria and chloroplasts of plants and fungi)
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Self-Splicing Group II Introns Provide Clues to
the Evolution of snRNAs
Even though the precise sequences of group II introns are not
highly conserved, they fold into conserved, complex secondary
structure containing numerous stem loops
The chemistry of self splicing by a group II intron is similar to that
found in pre-mRNA. This observation led to a hypothesis that
“snRNA function analogously to the stem-loop in the secondary
structure of group II introns”
According to this hypothesis, one expect to see the 3D structures
presented in the previous slide
Introns in ancient pre-mRNAs evolved from group II self-splicing
introns through progressive loss of internal RNA structures, which
concurrently evolved into trans-acting snRNA that perform the
same functions
Support for this type of evolutionary model comes from
experiments with group II intron mutants in which domain V and I
are deleted. The resulting mutant fail to perform self-splicing
Maturase enzyme may function to stabilize the 3-D structure for
self-splicing of group II introns in vivo
Nuclear Exonucleases Degrade RNA That Is
Processed Out of Pre-mRNA
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The spliced introns are degraded by exonucleases from 5’
or 3’ end.
The 2’,5’-phosphodieaster bond in the newly spliced
intron is excised to linear structure by a debranching
enzyme. The predominant decay pathway of the linear
RNA molecule is 3’ to 5’ by 11 exonucleases that
associate with one another in a large protein complex
called the “exosome”
Other protein in the exosome is RNA helicase that disrupt
baser pairing and protein RNA interactions
Exosome also functions to degrade improper processed
or polyadenylated pre-mRNA
Nuclear cap-binding complex: protein that bind to 5’cap
to prevent the cap been destroyed by exonulceases
Chain Elongation by RNA Pol II Is Coupled to the
Process of RNA-Processing Factors
• The carboxyl-terminal domain (CTD) of RNA Pol II is composed of multiple
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repeats of a seven residue sequence. When fully extended, the CTD domain
in yeast enzyme for instance is about 65 nm long
The CTD of human RNA Pol II is about twice as long
The long CTD allows multiple proteins to associate with a single RNA Pol II
molecule
For instance, enzyme that adds the 5’ cap to the nascent transcription
initiation as well as RNA splicing and polyadenylation factors are associated
with phosphorylated CTD. As a consequence, these processing factors are
present at high local concentrations when splice sites and poly(A) signals
are transcribed by the polymerase, enhancing the rate and specificity of
RNA processing. Deletion of CTD will reduce transcription and processing
of RNA
The association of RNA splicing factors with the phosphorylated CTD
stimulates transcription elongation
Coupling of Transcription and RNA Processing
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Transcription and RNA
processing are coupled
in the nucleus:
 Capping
 Release pausing
 Splicing
 Polyadenylation
Recruiting of these
various factors is
closely coupled to the
phosphorylation of the
CTD of RNA polymerase
II (see next slide for
details)
CE: capping enzyme;
SC: splicing complex;
PC: polyadenylation
complex; ph:
phosphorylation
Methylation of Arginine at H3 Stimulates RNA Splicing
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Trimethylation of the arginine at
position 4 in histone H3 not
only results in a more open
chromatin structure but also
stimulates transcriptional
elongation and enhances RNA
splicing
Both the CBC and the CPSF
polyadenylation complex can
interact with splicesome (S), so
linking together these different
post-transcription events
Splicing Facilitate Transport of mRNA to cytoplasm
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EJC: exon junction
complex, recruiting
several protein complex
for mRNA transport
Splicing can occur
during or after
transcription.
The transcription and
splicing machineries are
physically and
functionally integrated.
Splicing is connected to
mRNA export and
stability control.
Exon junction complex
(EJC) – A protein
complex that assembles
at exon–exon junctions
during splicing and
assists in RNA transport,
localization, and
degradation.
REF/Aly, a key protein
mediating mRNA
transport by interacting
with TAP (Mex67p)
The EJC Complex
Couples Splicing with
NMD
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Splicing in the nucleus can
influence mRNA translation in
the cytoplasm.
Some aberrant mRNA in the
cytoplasm that still has EJC
associate with it, the EJC can
recruit Upf that promotes
decapping enzyme to remove
CAP from the mRNA and
leading degradation of the
mRNA
Nonsense-mediated mRNA
decay (NMD) – A pathway that
degrades an mRNA that has a
nonsense mutation prior to
the last exon.
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Regulation of Pre-mRNA Processing
Macromolecular Transport Across
the Nuclear Envelop
Regulation of Pre-mRNA Processing
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Alternative splicing is the principle mechanism for
regulating mRNA processing
 By comparison of the genomic sequences of genes and the
sequences of the expressed sequence tags (ESTs) of cDNAs
reveals that several genes have complex transcription units, and
capable of producing several different mRNAs by different
combinations of exons
 More than 60 % of all transcription units in human genome are
complex
 Although cleavage at alternative poly(A) sites of pre-mRNA are
known example, alternative splicing of different exons is the
more common mechanism for expressing different protein from
one complex transcription unit
 Such alternative processing pathways are usually regulated in a
cell-type specific or developmental stage-specific manner.
Example: different fibronectin isoforms are produced in
fibroblasts and hepatocytes
Different Mode of Alternative Splicing
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Specific exons or
exonic
sequences may
be excluded or
included in the
mRNA products
by using
alternative
splicing sites.
Alternative
splicing
contributes to
structural and
functional
diversity of gene
products.
Effect of Alternative Splicing on Gene Expression (I)
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Alternative splicing can affect gene expression in the cell at least
in two ways:
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Create structural diversity of gene products by including or omitting
some coding sequences or creating alternative reading frames for a
protein of the gene. Example: CaMKIId gene encoding kinases
Differentiatial splicing of the
pre-mRNA for kinase results
in production of three
different kinases localized in
different locations in the cells
and kinasing different
substrates
 Another example is that alternatively spliced products may exhibit
opposite functions. This example applies to all genes involved in
apoptosis; one form promote apoptosis and the other form protect
cells from apoptosis
Alternative Splicing of Primary
Transcripts Where Both 5’ and 3’ Ends
of Transcripts Are Identical
 In muscle troponin T gene, the same
precursor mRNA can be spliced in up to 64
different ways in different muscle cell
types due to the presence of tissuespecific splicing factors
 The indication of the presence of tissue
specific splicing factors for differential
removal of exons 4-8 in different muscle
cells comes from the following experiment.
When muscle troponin T gene is
expressed in non-muscle cell types, exons
4-8 are completely removed and resulting
in only one mature mRNA.
Expression of Dscam Isoforms in Drosophila
Retinal Neurons
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The most extreme example of regulated alternative RNA
processing is the expression of Dscam gene in Drsophila
Dscam gene encodes a set of proteins in the neuron of
Drosophila
Mutations in this gene interfere with the normal connections
made by the axons of the retinal neurons with neurons in a
specific region of the brain
There are 95 alternatively spliced exons that could be spliced to
generate 38,000 possible isoforms
These results raise the possibility that the expression of
different Dascam isoforms through regulated RNA splicing
helps to specify the tens of thousands of different specific
synaptic connections made between retinal and brain neurons
In other words, correct wiring of neurons in the brain may
depend on regulated RNA splicing
Expression of Ca++-Activated K+ Channel Isoforms mRNAs
in Vertebrate Hair Cells Is Another Example
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In the inner ear of vertebrates,
individual “hair cells” which are
ciliated neurons, respond most
strongly to a specific frequency of
sound
In birds and reptiles, the turning of
hair cells is affected by the
opening of K+ channel in response
to Ca++ concentration changes.
The channel opens determine the
frequency with which the
membrane potential oscilates, and
hence the frequency to which the
cell is turned
Slo gene controls the channel,
which is expressed in multiple,
alternatively spliced mRNAs.
Suggested Reading List Vi:
 Distribution of Ca++-activated K+
chennel isoforms along the
tonotopic gradient
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Slo proteins encoded by different alternative spliced mRNAs open Ca++activated K+ channel at different Ca++ concentrations
Hair cells with different response frequencies express different Slo
channel protein depending on their position along the length of the
cochlea
There are 8 regions on the slo mRNA where alternative exons are utilized,
permitting the expression of 576 possible isoforms
RT-PCR analysis of sol mRNAs from individual hair cells has shown that
each hair cell expresses a mixture of different alternative spliced sol
mRNAs with different forms predominating in different cells according to
their position along the cochlea
In rat, the splicing at one of the alternative splice sites in the slo premRNA is suppressed when a specific protein kinase is activated by
depolarization of the neurons. This observation suggests that a splicing
repressor specific for this site may be activated when it is
phosphorylated by this protein kinase
These observations suggest that modification of splicing factors may
play a significant role in modulating neuron function
Suggested reading List IV:
– Distribution of Ca++-Activated K+ Channel Isoforms along the Tonotopic
Gradient
Effect of Alternative Splicing on Gene Expression (II)
 Alternative splicing may affect various properties of the mRNA by
including or omitting certain regulatory RNA elements, which may
significantly alter the half-life of the mRNA. This form of the splicing
is caused by the presence of a splicing factor
 This figure shows models by which an alternative splicing factor can
affect splicing by binding to a cis-acting element. In (a), the factor
acts by promoting the use of the weaker of the two potential splicing
sites while in (b) it acts by inhibiting use of the stronger of the two
sites so that other, weaker, site is used.
 The sex determination in Drosophila is the best characterized
example
Example: Regulation of Alternative RNA Splicing
Leading to Sexual Differentiation in Drosophila
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The earliest example of regulated alternative splicing of pre-mRNA
came from studies of sexual differentiation in Drosophila
Sxl protein, encoded by sex-lethal gene, is produced in female
Drosophila by a promoter only functions in female embryos. Later
in embryonic development, this female-specific promoter is turned
off, and a promoter is tuned on in both male and female embryos.
In the absence of Sxl protein in male embryos, the sex-lethal premRNA is processed into a mature mRNA with a stop codon in the
sequence and thus produce no functional Sxl protein
Sxl protein directs splicing of sex-lethal pre-mRNA to produce
functional Sxl mRNA
Cascade of Regulated Splicing that Controls Sex
Determination via Expression of Sex-Lethal (sxl) Transformer
(tra) and Double-Sex (dsx) Genes in Drosophila
•
•
•
•
•
•
The earliest example of regulated differentiation splicing of premRNA come from studies of Drosophila sexual differentiation
Two proteins were found to regulate differential splicing of premRNA, one of which is Sxl protein and the other is Tra protein
Sxl protein regulates alternative RNA splicing of the Sxl mRNA in
early embryonic development of females but not in males
Sxl protein also regulate splicing of transformer gene pre-mRNA
in females by the similar mechanism and the Tra protein is
produced
In female embryos, Tra protein and two other constitutive
expressed proteins, Rbp1 and Tra2, directs splicing of exon 3 to
exon 4 of the double-sex pre-mRNA and also promotes
cleavage/polyadenylation at the alternative poly (A) site at the 3’
end of the exon 4 and thus producing female specific Dsx protein
In male embryos, due to the absence of Tra protein, a different Dsx
protein is produced which functions as a repressor to inhibit the
expression of genes essential for female development in male
embryos. Conversely female Dsx protein inhibits the expression
of genes for male development in female embryos
Splicing Can Be Regulated by Exonic and
Intronic Splicing Enhancers and Silencers
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•
•
•
Alternative splicing is often associated with weak splice sites
Sequences surrounding alternative exons are often more
evolutionarily conserved than sequences flanking constitutive
exons
Specific exonic and intronic sequences can enhance or suppress
splice site selection
SR proteins are the known proteins binding to ESE; hnRNP A/B
proteins are known to bind to ESS; RBP proteins are known to
bind to ISS or ISE
The Nova and Fox Families of RNA Binding Proteins
•
•
•
•
The effect of splicing
enhancers and silencers
is mediated by sequencespecific RNA binding
proteins, many of which
may be developmentally
regulated and/or
expressed in a tissuespecific manner.
The rate of transcription can directly affect the outcome of
alternative splicing.
Nova and Fox families of RNA binding proteins can promote or
suppress splice site selection in a context dependent fashion.
Binding of Nova to exon or flanking upstream introns inhibits the
inclusion of alternative exon while Nova binding to the
downstream flanking intron sequences promotes the inclusion of
the alternative exon.
Fox binding to the upstream intron inhibits the inclusion of the
alternative exon and vice versa
Splicing Repressors and Activators Control
Splicing at Alternative Sites
•
•
Sxl protein prevents splicing, causing exons to be skipped
whereas Tra promotes splicing. Therefore, Sxl protein is
considered as a repressor for splicing and Tra protein is
considered as an activator for splicing
In the processing of the fibronectin pre-mRNA, Sxl like repressor
could be expressed in the human hepatocytes to bind to the splice
sties of the EIIIA and EIIIB exons in the fibronectin pre-mRNA,
causing them to be skipped during splicing. On the other hand, a
Tra-like protein could be expressed in the fibroblasts and resulting
in inclusion of EIIIA and EIIIB in the fibronectin mRNA
RNA Editing Alters the Sequences of the Pre-mRNAs
•
•
•
•
RNA editing: the sequence of the pre-mRNA is altered; and as the result, the
sequence of the corresponding mature mRNA differs from the exons
encoding it in the genomic DNA
RNA editing is widespread in the mitochondria of protozoans and plants, and
also in chloroplasts
In higher vertebrates, RNA editing is much rarer, and mostly in single base
changes
ApoB gene in mammals is one of the example of RNA editing. In the liver,
ApoB gene is transcribed into a longer mRNA which encodes a protein with
two domains (green domain binds to lipid and the orange domain binds LDL
receptor). In the intestine, the CAA codon of ApoB is edited to UAA [a stop
codon] so the protein [ApoB-48] can bind lipid but not with LDL receptor
RNA Editing Involves a Change from an A to an I
 Besides C- to U- editing, A- to I- editing has also been observed in
human
 An editing of A- to I- residue in the transcript of a receptor for the
excitatory amino acid glutamate in neuronal cells results in
encoding an arginine rather than glutamine, leading to the
receptor permeable to calcium
 Animals with this editing yet lacking ADAR2 (adenosine
deaminase enzyme) will result in death
Effect of A- to I- RNA Editing and RNA Splicing
 A- to I- editing of ADAR2 mRNA can also affect the splicing of this
mRNA as well as the encoded protein
 Another effect of A- to I- editing can also target micro RNA
(miRNA) by affecting it binding to the target RNA (The function of
miRNA will be discussed later)
Effect of A- to I- Editing Affects the Function of miRN A
 A- to I- editing in miRNA376
will result in a tissue specific
effect in mice
 It results in inhibiting a
different set of genes in
kidney but not in the liver
 In conclusion, the existence
of two distinct types of
editing enzymes, adenosine
and cytidine deaminases, in
mammalian cells each with
multiple substrates indicates
that this process represents
a widely used mechanism
which like alternative
splicing that can produce
related but distinct proteins
from the same gene
Macromolecular Transport Across the Nuclear Envelop
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•
•
•
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Messenger Ribonuclear
Protein Complex (mRNP):
mature mRNA complexed
with specific hnRNP, the
complex will be transported
out of the nucleus
The nucleus is separated by a
double membrane (nulcear
envelope) from the cytoplasm
Mature mRNPs, tRNAs,
ribosomal subunits are
transported out of the nucleus
through nuclear pores, and
transport of nuclear proteins
from cytoplasm into nucleus
is also through the same route
Small molecules and globular proteins >60 kDa can diffuse directly through
the pores
Large proteins and ribonucleoproteins are transported by the aid of
transporters
•
•
Large and Small Molecules Enter or Leave the
Nucleus via Nuclear Pore Complex
Nuclear Pore Complexs (NPCs): Large symmetrical structures
composed multiple copies of approximately 30 different nuclear
proteins
Under EM, the nuclear pore complexes showed a octagonal,
membrane-embeded structure from which eight (~100 nm-long)
filaments extend into the nucleoplasm. The distal ends of these
filaments are joined by the terminal ring forming a nuclear basket
•
•
The membrane
embeded protein is
also attached directly
to nuclear lamina
The cytoplasmic
filaments extend from
the cytoplasmic side of
the NPC to the cytosol
•
•
•
•
•
Four proteins are required for transporting macromolecules into
the nucleus have been purified. These proteins are: Ran,
nuclear transport factor 2 (NTF2), importin a, importing b
Ran is a monomeric G protein, existing in two conformations,
one can complexe with GTP and another one with GDP
Importin a and importing b form a heterodimeric nuclear-import
receptor, the a submit bind to the NLS (nuclear localization
signal) and the b-submit binds to a class of nuclearproteins (FGnucleoproteins). These nucleoproteins line the channel of the
nuclear pore complex and are also found in the nuclear basket
and the cytoplasmic filaments.
FG-nucleoproteins contain multiple short repeats rich in
phenylalanin (F) and glycine (G) residues
Recently several importin b analogs have been identified which
can transport proteins with other class of NLS into nucleus
without the need to bind with importin a
FG-Domains of FG-Nucleoproteins
The FG-domains
of FGnucleoproteins
The FG-repeats are
thought to associate
with each other
reversibly and rapidly
forming a constantly rearranging molecular
sieve that allows
diffusion of small watersoluble molecules
through it
•
•
•
mRNA Exporter Helps Export of mRNP’s from
the Nucleus to Cytoplasm
Temperature sensitive mutation studies in yeast revealed that a
heterodimeric mRNA-exporter is required for export of mRNPs out of
nucleus
mRNP exporter (TAP) is a heterodimer consisting of a large submit (NXF1)
and a small submit (NXT1). TAP binds to mRNP through association with
RNA and other proteins in the mRNP (REF, RNA export factor)
The TAP/NXT1 mRNP also associate with SR proteins (splicing factors)
that will direct both splicing and transport
Small submit of mRNA-exporter is
homologous to NTF2 (nuclear
transport factor 2), and interacts with
a region of the larger submit that also
shares homology to NTF2. Together,
they form a domain that interacts
with FG repeats in FG-nucleoproteins
similar to NTF2 which is a dimer
A C-terminal domain of the mRNA
exporter also interacts with FGrepeats
•
•
Remodeling of mRNPs During Nucleat Export
•
•
Some mRNP proteins dissociate from the nuclear RNP complex before
export through an NPC while the others are exported to the cytoplasm and
dissociated from the mRNP and shuffled back to nucleus
These proteins include NXF1/NXT1 mPNP exporter, cap-binding complex
(CBC), and PABPII
Reversible Phosphorylation and Direction of
mRNP Nuclear Export
• Results of recent studies indicated
that direction of mRNP export
from the nucleus into the
cytoplasm is controlled by
phosphorylation and
dephosphorylation of mRNP
adaptor proteins such as REF
In yeast, SR protein (Npl3)
functions as an adaptor protein
that promotes binding of the yeast
mRNP exporter. Phosphorylation
and dephosphorylation are
involved
1. Phosphorlated NpI3 binds to
nascent pre-mRNA
2. Following polyadenylation, the
NpI3 is dephosphorylated and
promoting the binding of NXF1/NXT1 the mRNP complex
3. The mRNP exporter allows transport of mRNP
4. Sky (cytoplasmic kinase) phosphorylates NpI3 in the cytoplasm
5. Dissociaation of NXF1/NXT1 and NpI3 from the mRNP and return to nucleus
•
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•
•
•
•
Other Proteins Assist mRNP Transport
•
Some mRNP proteins assist binding of mRNA with
mRNA-exporter





SR proteins
Exon junction complex
Nuclear cap binding complex
Poly (A)-binding protein (PABPI)
Other RNA-binding protein
Nuclear Export of Balbiani Ring mRNPs
•
•
The salivary glands of larvae of the Chironomous
tentans has large RNA containing chromosome puffs
called Balbiani Ring containing RNA-protein complex.
These giant RNPs contain mRNA (75 kb) of glue protein
After processing, the pre-mRNA in Balbiani ring in the
form of RNP migrate through the nuclear pores
Formation of Hetergenous hnRNPs and
Export of mRNPs
Single chromatin transcription loop
containg assembled Balbiani ring
RNP
The schematic drawing of the
biogenesis of hnRNP
Model for the Transport of BR mRNPs Through
the Nuclear Pore Complex (NCP)
Transport of the Balbiani Ring RNP across the Nuclear Pore Complex (NPC)
But pre-mRNAs in spliceosomes are not exported from
the nucleus
Fusion of a Nuclear Localization Signal (NLS) to a
Cytoplasmic Protein Causes the Protein to Enter
the Cell Nucleus
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•
•
•
Nuclear-localization
signal (NLS) is the
sequence found in large
T-antigen of simian virus
(SV40)
The particular sequence
is Pro-Lys-Lys-Arg-LysVal at the C-terminal end
of the large T-antigen
Fusion of the NLS
sequence to other
cytoplasmic protein will
result in transport of the
protein to the nucleus
Unlike the NLS of T-antigen, an NLS in the hnRNP A1 protein is
hydrophobic
Mechanism for Nuclear Import of “Cargo” Proteins
The details of the
transport are
described in next
slide
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Free monomeric importin forms complex with the cargo protein
by binding to the NLS of the cargo in the cytoplasm
The cargo-importin complex enters the nucleus through the
nuclear pore complex as the importin binds to FG-proteins
In the nucleoplasm, the importin binds to Ram-GTP to form RanGTP-importin complex and the cargo is released in the
nucleoplasm
The Ran-GTP-importin complex diffuse through the NPC to the
cytoplasm
In the cytoplasm, importin interacts with a specific GTPaseaccelerateing protein (Rn-GAP) that is a component of the NPC
cytoplasmic filaments, This stimulates Ran to hydrolyze its
bound GTP to GDP causing Ran has low affinity to importin
The importin will participate in another cycle of transporting new
cargo, while the Ran-GDP will return to nucleus by nuclear
transport factor 2 (NTF2) and a guanine nucleotide-excchange
factor (GEF) will cause release of GDP and rebinding to GTP
The entire transport is driven by a facilitated diffusion model
Ran-Dependent Nuclear Transport
•
•
•
NES: Nuclear export
signal on the cargo
Exportin (nuclear
export receptor):
Nuclear protein
binding to cargo
directed for export
from nucleus to
cytoplasm
Export of molecule
out of the nuclear
requires Ran-GTP
Ran-Independent Nuclear Transport
•
•
•
Most mRNAs are
transported from
the nucleus by a
Ran-independent
mechanism
mRNA exporter: a
heterdimer of
TAP/NXT1
This form of
transport does
not require RanGTP
•
•
Cytoplasmic mechanisms of posttranscriptional regulation
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

Regulatory RNAs
Localization of mRNAs and their stability
Global regulation fo protein synthesis
Processing of rRNA and tRNA
Regulator RNA
•
•
RNA functions as a regulator by forming a region of secondary
structure (either inter- or intramolecular) that changes the
properties of a target sequence.
The formation of double stranded structure between regulator
RNA and the target mRNA will have two consequences:
 Formation of the double-helical
structure may itself be
sufficient for regulatory
purposes (preventing
translation or degradation by
ribonucleases)
 Duplex formation may be
important because it
sequesters a region of the
target RNA that would
otherwise participate in some
alternative secondary structure
A Riboswitch Can Alter Its Structure
According to Its Environment
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•
•
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•
A riboswitch is an RNA whose activity
is controlled by the metabolite
product or another small ligand (a
ligand is any molecule that binds to
another).
A riboswitch may be a ribozyme.
Aptamer – An RNA domain that binds
a small molecule; this can result in a
conformation change in the RNA.
An enzyme encoded by glms which
catalyzes the synthesis of GlcN6P
(glucosamine 6 phosphate) from
fructose-6-phosphate
GlcN6P binds to the ribozyme
sequence in the 5’ UTR leading to
cleavage of the mRNA
Noncoding RNAs Can Be Used to Regulate
Gene Expression
•
•
•
•
•
•
Vast tracts of the eukaryotic
genome are transcribed.
Antisense gene – A gene that
codes for an antisense RNA that
has a complementary sequence to
an RNA that is its target.
Antisense RNA – RNA that has a
complementary sequence to an
RNA that is its target.
Therefore, antisense gene can be used to turn off the expression
of a gene at will
Nested genes: Genes located within the introns of other genes.
About 10-15% of genes have nested genes. If these genes are
transcribed from the opposite strand, antisense RNAs are
produced. The head to head arrangement of a nested gene will
lead to transcriptional interference (TI).
Transcriptional interference is emerging as a significant
mechanism of transcriptional regulation, is the origin of the
interference RNA
Antisense RNA Regulates Gene Expression.
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•
•
•
•
Recent studies showed that
about 70% the human genes
produce anti-sense RNA.
This patent various with the
cell types. In addition, DNA
in the intergenic regions are
also transcribed. These RNA
products can result in noncoding RNAs with regulatory
function
In yeast, gene PHO84 is regulated in part by a class of non-coding RNAs
named “cryptic unstable transcripts” (CUTs) transcribed from a promoter
on the non-coding strand of PHO84
Under the normal condition, this transcript is unstable and rapidly
degraded by TRAMP and exosome RNase complex
In the absence of degradation or in aging cells, the anti-sense RNA
persists. It recruits histone deascetylase to remove acetyl group from the
acetylated histone and thus causes remodeling of chromatin and turns off
the gene activity
In human, PROMPTs (promoter upstream transcripts) may have the same
function
TRAMP Complex
TRAMP complex (Trf4/Air2/Mtr4p Polyadenylation complex) is a protein
complex consisting of the RNA helicase (Mtr4), a poly(A) polymerase
(PAP) (either Trf4 or Trf5) and a zinc knuckle protein (either Air1 or
Air2). The TRAMP complex interacts with the Exosome complex in the
nucleus of eukaryotic cells and is involved in the 3’ end processing of
ribosomal and snoRNAs (small nuclolar RNA)
•
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•
Bacteria Contain Regulator RNAs
Bacteria contain up to hundreds of genes encoding RNA
molecules ranging from 50 to 200 bp with regulatory functions.
These RNA molecules are called regulator RNAs (sRNAs)
Some of the sRNAs are common for several genes and others
are specific to individual genes
All sRNAs are imperfect anti-sense RNA molecules
sRNAs function to (i) prevent transcription of the gene; (ii) affect
processing of its RNA product; (iii) affect translation of the
mRNA; and (iv) affect the stability of the mRNA by the formation
of RNA-RNA duplex
Example: Oxidative stress in E. coli is an example


When E coli is exposed to reactive oxygen species, the expression
of antioxidant defense genes are induced. Hydrogen peroxide
activates the transcription of OxyR which activates the transcription
of several inducible genes including OxyS which encodes sRNA
OxyS gene product is a small RNA of 109 bases long. It is a transregulator that repress the translation of fihA mRNA
Effect of OxyS RNA on Translation of FihA mRNA
Duplex formation between
fihA mRNA and oxyS RNA
•
•
•
•
OxyS RNA can also regulate rpoS, a gene coding for an alternative
sigma factor. RpoS gene can also be positively regulated by two
sRNA (dsrA and rprA). These three sRNAs are the global
regulators that coordinate responses to environmental conditions
Several of the sRNAs are bound by the protein Hfq, which
increases their effectiveness
The oxyS sRNA activates or represses expression of >10 loci at
the post-transcriptional level
We just to realize that many small RNAs in bacteria possess
activities in controlling processes in the life cycle. Tandem
repeats like CRISPR can be transcribed into powerful antiviral
RNAs. The example is shown in next slide
The CRISPRs System in E. coli
The CRISPRs (clusters
of regulatory
interspersed short
palindromic repeats)
locus in E. coli is
transcribed into a large
precursor RNA, which
is processed by the
cascade protein
complex into short
fragments that contain
unique spacers
identical to sequences
in the phage DNA.
Assisted by a protein,
Cas3, these small CRISP RNAs block the phage infection cycle.
These mechanisms offer powerful approaches for turning off genes at
will.
•
Cytoplasmic Mechanisms of Posttranscriptional Control (I)
Micro RNA:
 was discovered in the nematode (C. elegans) during analysis of lin-4
and let-7 genes
 Cloning and nucleotide sequence analysis revealed that lin-4 and let-7
do not encode any protein product, but encoding RNAs of 21 and 22
nucleotides long, respectively. lin-4 RNA hybridizes to the 3’untranslated region of lin-14 mRNA and lin-28 RNA and degrades the
mRNA
 About 100 different miRNA have been found in C. elegans and at least
as many or more found in human. These miRNAs are used to degrade
many mRNAs. MicroRNAs are transcribed by Pol II
 All miRNA precursors are about 70 nucleotides long, and are
processed by a ribonuclease (Dicer) to produce mature miRNAs of 2122 bases
 Base pairing between the miRNA and the 3’-untranslated region of an
mRNA does not have to be 100% complementary. This differentiates
it from the RNA interference. In human, more than 1,000 different
miRNA are produced
 Reading List VI:
 (i) Micro RNA
 (ii) Micro RNA controlling cell division – differentiation and cell death
RNAi Inhibits Transcription
•
•
•
In an attempt to inhibit the translation of an mRNA in C. elgans by
microinjecting single stranded antisense RNA, an unexpected
result was found in the control experiment. In this control
experiment, perfectly base paired double stranded RNA of
several hundred bases long was microinjected into C. elegans,
more effective inhibition was observed. Similar results were
observed in plant system. This led to the discovery of siRNA
(RNAi)
Further studies revealed there are large number of double
stranded transcribed from non-protein coding genes in the
nucleus. These RNA molecules are processed into 21-23 bases
long siRNA by enzyme systems similar to the processing of
miRNA
Nuclear proteins homologous to the cytoplasmic “Dicer” and
“Argonatue protein generate nuclear siRNA complexes
composed of different proteins from cytoplasmic RISC
complexes. These nuclear siRNA complexes are thought to
targeted to specific gene by base paring with nascent pre-mRNAs.
This interaction induces the methylation of histone H3 at lysine 9,
generating a binding site for HP1 proteins and the subsequent
assembly of heterochromatin
Cytoplasmic Mechanisms of Posttranscriptional Control (II)
•
RNA interference:
 RNA interference induces degradation of mRNAs with sequence
complementary to double-stranded RNAs
 RNA interference was discovered from initial attempts to inhibit the
translation of an mRNA by microinjecting a RNA with complementary
sequence (antisense inhibition). In the control experiment, scientists
found that perfectly base-paired double-stranded RNA of a few hundred
pairs long was much more effective in inhibiting the expression of the
gene than the anti-sense RNA alone
 It was found that as long as one of the strand the ds-RNA is
complementary to the mRNA sequence, it can effectively inhibit the
mRNA
 Further studies revealed that the long double-stranded interfering RNA
was processed into short dsRNA (siRNA) of 21-23 nucleotides double
stranded region with 3’ single stranded and Dicer ribonuclease is
required for the processing
 A Nobel Prize was awarded to Andrew Fire and Craig C. Mello on
discovering RNAi in nematode worm
 Reading List (IV):
 (i) RNA interference; (ii) Ribo-genome: the big world of small RNAs; (iii) Nobel
lecture on RNAi by Mello; (iv) Nobel lecture on RNAi by Fire.
Model for mRNA Translational Repression and RNA
Interference Mediated by the RNA-Induced
Interference Complex (RISC)
•
•
The double-stranded
siRNA and miRNA are
further processed into a
multi-protein complex
containing only one
strand of RNA
This RNA-induced
silencing complex (RISC)
cleaves target RNAs that
is precisely
complementary to their
corresponding singlestranded siRNAs. These
complexes also appear to
function in the inhibition
of translation by miRNA
miRNA Processing
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•
•
•
•
•
This diagram shows the processing of
miR-1-1 miRNA
Pre-miRNA is transcribed by RNA
polymerase II
Nuclear double-stranded specific
endonuclease “Drosha” with its
partner double-stranded RNA binding
protein “DGCR8” make the initial
cleaves in the pri-miRNA to generate a
70-nucleotide pre-miRNA
The 70-nucleotide pre-miRNA is
transported to cytoplasm by nuclear
exporter
In the cytoplasm, the 70-nucleotide
pre-miRNA is processed by Dicer to
form miR-1-1 miRNA
One of the strands of miRNA is
incorporated into an RISC complex
with Argonaute protein
•
•
•
•
•
The binding of several RISC complexes to an mRNA inhibits
translation initiation
Binding of RISC complexes cause the bound mRNPs to
associate with dense cytoplasmic domains many times the size
of a ribosome called cytoplasmic RNA-processing bodies, Pbodies, the site of RNA degradation
About 1000 different human miRNAs have been observed,
many of them expressed only in specific cell types
miR-133, a specific miRNA is induced when myoblasts
differentiate into muscle cells. miR-113 suppresses the
translation of PTB, a regulatory splicing factor that functions
similar to Sxl in Drosophila
Knocking out of “Dicer” gene eliminates the generation of
miRNA in mammals. In mouse knocking out Dicer gene will
result in early embryonic death. If it is in limb primordia, it will
result in abnormal limb development
Base Pairing with Target RNAs
Distinguishes miRNA and siRNA
miRNA Function in Limb Development
Mouse embryos
Methods of Generating RNAi
Cytoplasmic Polyadenylation Promotes
Translation in Some mRNAs
•
•
5’ and 3’ untranslated
sequence in the mRNA
could also contain
regulatory functions
In immature oocytes, many
mRNA containing U rich
and short poly (A) are
inactive for translation.
These mRNAs have to be
polyadenylated in the
cytoplasm before they can
be activated for translation.
•The model used to explain the activation of these mRNAs is presented above.
•CPE: cytoplasmic polyadenylation element; CPEB: CPE-binding protein
•PABPI: poly (A) binding protein I; CPSF: cleavage & polyadenylation
specificity factor; PAP: poly (A) polymerase
Pathways of Degradation of Eukaryotic mRNA
•
•
•
•
The concentrations of an mRNA is a function of its synthesis and
degradation
Steady state level of mRNA: can be determined by extracting mRNA
from a tissue and determine the level of the mRNA by various
methods
Bacterial mRNA is very labile, while mRNA from multi-cellular
organisms is very stable
Cytoplasmic mRNA is degraded by any one of the mechanisms
listed above
Protein Synthesis Can Be Globally Regulated
• Synthesis of cellular proteins can be regulated via post-
•
translational modification of initiation factors or ribosomal
proteins via phosphorylation
TOR Pathway:
•
The pathway of mTOR is summarized in next slide
 Rapamycin is an antibiotics produced by Streptomyces bacteria
which is able to suppress immune response in organ transplantation
patients.
 TOR (target of rapamycin) was identified in yeast by isolating yeast
mutants resistant rapamycin inhibition of cell growth. TOR is a large
protein kinase (2400 a.a.) that regulate several cellular processes in
yeast cells in response to nutritional status. Metazoan TOR (mTOR)
has also been isolated. It works similar to that of yeast TOR
 Active mTOR stimulates the overall protein synthesis by
phosphorylating two proteins that regulate translation directly
mTOR also activates transcription factors that control expression of
ribosomal components, tRNAs, and translation factors, further activating
cell growth with developmental programs as well as nutritional status
mTOR Pathway
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•
•
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•
Active mTOR: mTOR
complexes with RhebGTP
Inactive mTOR: mTOR
complexes with RhebGDP
TCS1/TCS2-Rheb-GAP
is phosphrylated by
AMP-kinase
Rheb-GAP: RhebGTPase activating
protein which can be
activated by AMPK
TCS proteins were
identified from human
disease, tuberous
sclerosis complex,
forming benign tumors
mTOR Pathway (Continued)
 The first step of translation of eukaryotic mRNA is binding of the
eIF4 initiation complex to the 5’ cap via eIF4E cap-binding submit.
The concentration of active eIF4E is regulated by a small family
of homologous eIF4E-binding proteins (4E-BPs) that inhibits the
interaction of eIF4E with mRNA 5’-cap
 When 4E-BPs are phosphorylated by mTOR, 4E-BPs release
eIF4E and resulted in stimulation of translation initiation.
 mTOR also phosphorylates and activates another protein kinase
that phosphorylates small ribosomal submit protein S6 (S6K),
leading to further increase in the rate of protein synthesis
 mTOR stimulates the translation of mRNAs with a string of
pyrimidine in the 5’ untranslated region (TOP mRNAs for tract of
oligopyrimidine) that encode ribosomal proteins and translation
elongation factors
 mTOR also activates RNA polymerase I transcription factor,
TIF1A, activates transcription by RNA polymerase III, and
activates two polymerase II activators that stimulate ribosomal
genes
mTOR Pathway (Continued)
 mTOR stimulates processing of the rRNA precursor
 mTOR activity is regulated by a monomeric small G protein in
the Ras protein family called Rheb. Rheb-GTP activates mTOR
kinase. Rheb is in turn regulated by TSC1 and TSC2
heterodimer by hydrolyzing GTP to GDP
 mTOR activity is also regulated in response to nutritional status.
When energy from nutrients is not sufficient for cell growth, the
resulting fall of ATP/AMP ratio is detected by AMP-kinase and
phosphorylates TSC1/TSC2 to stimulate Rheb-GAP activity and
leading to inhibition of mTOR kinase activity
 In addition to regulating the global rate of cellular protein
synthesis and production of ribosome, tRNAs, and translation
factors, mTOR also regulates macroautophagy (degradation of
cellular components).
eIF2 Kinase
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•
•
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eIF2 kinases also regulate global rate of protein synthesis
eIF2 is a trimeric G protein existing in eIF2-GTP or eIF2-GDP
form, which can bind to charged initiator tRNA and in
association with small submit of ribosome
Phosphorylation of eIF2 a-submit at a specific serine moiety
resulted tight binding of eIF2 to GTP leading to failure of
converting GTP to GDP, leading to inhibition of protein
synthesis
Cellular stress will lead to the same results
Human cells contain 4 eIF2 kinases that phosphorylate the
same inhibitory eIF2 a-serine. Each of these is regulated by a
different type of cellular stress
Iron-Dependent Regulation of mRNA Translation
and Degradation
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Sequence –specific RNA-binding proteins
control specific mRNA translation
Transferrin binds ingested iron in order to
transfer into cells
Iron-transferrin complex binds to transferrin
receptor residing on the cell membrane and
the complex is transported into the cell by
receptor-mediated endocytosis
At low intracellular concentrations of iron,
the IRE-BP (iron response element-binding
protein) is active which can bind the stem
loop of the AU-rich region and thus
preventing the transferrin mRNA been
degraded
At high concentration of iron, IRE-BP is
inactive and fails to bind to the stem loop of
the AU-rich region of transferrin mRNA, thus
no translation of ferritin mRNA
IRE-BP also regulate TfR (transferrin
receptor) mRNA by a similar mechanism
Nonsense-Mediated Decay and Other
mRNA Surveillance
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There are several mechanism preventing improperly
processed mRNA been translated into protein
products
mRNA surveillance is one of the mechanism to
remove improperly spliced mRNA in the nulceus
because the improperly spliced pre-mRNA is not
transported out of the nucleus and is degraded in the
nucleus
Nonsense-mediated decay of mRNA: Degrades mRNA
with one or more exons have been skipped. Exonjunction complex may be responsible for initiating
nonsense-mediated decay of mRNA
Localization of mRNAs Permits Production of Proteins at
Specific Regions within the Cytoplasm (I)
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For functional need some of the cellular proteins are
localized in specific locations in the cell
This is achieved by: transport the synthesized mRNA to
the desired location or by distribution of mRNA in the
desired location
A well-documented example of mRNA localization occurs
in mammalian myoblasts (muscle precursor cells). b-actin
mRNA is accumulated in the lamellipodia of the myoblasts
When myoblasts fuse into myotube, expression of b-actin
mRNA is repressed and expression of a-actin is seen in
the perinuclear region
Localization of mRNAs Permits Production of Proteins at
Specific Regions within the Cytoplasm (II)
This picture shows localization of neuronal mRNA to synapses. Sensory
neuron from sea slug. The motor neuron is visualized in blue and green color
is GFP-VAMP protein is in the synapse. The red color is the in situ
hybridization of the GFP-VAMP mRNA also shown in the synapse
Processing of Ribosomal RNA Precursor
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Approximately 80% of the total cellular RNA is rRNA and 15%
tRNA
Pre-rRNA genes are similar in all eukaryotes and function as
nucleolar organizers
Pre-rRNA is encoded by a single-type of pre-rRNA transcription
unit transcribed by RNA polymerase I
Pre-rRNA is 45 S which is processed into 28 S, 18 S and 5.8 S
rRNAs
There are several common properties among rRNA gene from
different species of eukaryotes:
 Pre-rRNA genes are arranged in tandem arrays separated by nontranscribed spacer regions ranging from 2.0-30 kb
 Genomic regions corresponding to the three mature rRNAs are arranged
in the same 5’ to 3’ order: 18S, 5.8S and 28S
 The transcribed regions are processed and degraded rapidly
EM of Pre-rRNA Transcription Units from the
Nucleolus of a Frog Oocytes
General Structure of Eukaryotic Pre-rRNA
Transcription Unit
Processing of Pre-rRNA
and Assembly of
Ribosomes in Higher
Eukaryotes
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Small nucleolar RNAs
(snoRNAs) assist the
processing of pre-rRNA and
assembling of ribosome
submits
Pre-rRNA binds to proteins to
form ribosomal
ribonucleoprotein particles
(pre-rRNPs)
Small Nucleolar RNAs
(snoRNAs) can form snoRNP
by hybridizing to pre-rRNA
5S RNA is transcribed from 5S
RNA gene by Pol III
Pre-tRNAs Undergo Cleavage, Base Modification
and Sometimes Protein-Catalized Splicing
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A 5’ sequence of
variable length is
precessed out
from the pre-tRNA
by ribonuclease P
(RNase P)
About 10% of the
bases in tRNA is
modified during
processing
Three types of modifications: (i) replacing U at the 3’ end with CCA; (ii)
addition of methyl and isomethyl groups to the heterocyclin ring of purine
base and methylation of the 2’-OH group in the ribose of any residue; (iii)
conversion of specific U to dihydro-U, Pseudo-U or ribothymidine
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Splicing of intron in pre-tRNA is different from
splicing of type I and type II introns in pre-rRNA and
splicing introns in pre-mRNA in the following three
ways:
 Splicing of pre-tRNA is catalyzed by proteins, not by RNAs
 A pre-tRNA intron is excised in one step that entails
simultaneous cleavage at both ends of the intron
 Hydrolysis of GTP and ATP is required to join the two tRNA
halves generated by cleavage on their side of the intron
Export of tRNA to cytoplasm require the mediation of
exportin-t
Generally speaking, tRNAs in the cytoplasm rarely
stay alone without in association with protein factors
such as aminoacyl synthetase, elongation factors
and ribosomes
Conclusion of Regulation of Gene Expression
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Regulation at transcriptional level:
 Regulation of initiation of transcription
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 Chromatin-mediated transcriptional control
 Activators and repressors interaction with transcription complex
Regulation at post-transcriptional level in the nucleus:
 Regulation of alternative splicing leading to production of
multiple isoforms of proteins
 Regulation of transport of mRNA into cytoplasm
Regulation at post-translational level in cytoplasm
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Micro RNAs
RNA intereference (RNAi or siRNA)
Cytoplasmic polyadenylation
mRNA degradation
Localization of mRNA in the cytoplasm
Assigned Reading
1.hnRNP complex
2.Heterogenous ribonuclear particles
3.R Loop Mapping
4.Nobel Lecture by Richard Sharp
5.Mapping of viral RNA with viral DNA
6.Cech’s Nobel Prize lecture
7.Hammerhead ribozyme
8.Distribution of Ca++-activated K+ chennel isoforms
along the tonotopic gradient
9.Micro RNA
10.Micro RNA controlling cell division – differentiation and
cell death