Transcript Trans - Wiley

Chapter 13:
RNA Processing and
Post-Transcriptional
Gene Regulation
The discovery of split genes along with
some other findings of recent years,
shows that the genetic apparatus of the
cell is more complex, more variable, and
more dynamic than any of us had
suspected.
Pierre Chambon, Scientific American (1981)
244:60
13.1 Introduction
• Scientists who study RNA have been faced
with more revolutionary and unexpected
discoveries in the past several decades than in
any other area of molecular biology.
• A recurring theme in processing events that
involve RNA cofactors is that the RNA is used
to provide specificity by complementary base
pairing.
13.2 The discovery of split
genes
• 1977: The discovery of split genes, first in
adenovirus and then in a number of
cellular genes.
• Example: The ovalbumin gene was
shown by R loop mapping to be split into
eight sets of sequences.
• 1978: Walter Gilbert coined the term
intron for the intervening sequences that
split genes.
• Until the early 1990s, the view was that
introns are “junk” that is excised and
degraded.
Now known that introns can be functional
• May contain transcriptional regulatory
elements.
• May code for small nucleolar RNAs and
microRNAs.
Modern definitions
• Introns are sequences that remain
physically separated after excision.
• Exons are sequences that are ligated
together after excision.
• RNA splicing is the process by which
introns are removed from a primary RNA
transcript at precisely defined splice
sites.
Intron-encoded small nucleolar RNAs
and “inside-out” genes
• snoRNAs mediate base modification of
ribosomal RNA (rRNA) and possibly
other RNAs.
• By complementary base pairing with the
rRNA, the snoRNAs recruit the
modification enzymes to their target
sites.
• Box C/D snoRNAs guide 2′-Omethylation.
• H/ACA snoRNAs direct pseudouridine
formation.
• In some “inside-out” genes, the introns
code for function and the exons are
degraded.
• In 1994, U22 snoRNA and seven other
snoRNAs were discovered within separate
introns of the U22 host gene.
13.3 Splicing occurs by a variety
of mechanisms
Five major classes of introns are
distinguished by their structure and
mechanisms of splicing
•
•
•
•
•
Autocatalytic group I introns
Autocatalytic group II introns
Archael introns
tRNA introns
Spliceosomal introns in nuclear pre-mRNA
Group I and group II self-splicing introns
• Large catalytic RNAs distinguished by
their structure and mechanisms of
splicing.
• Both types of introns have mobile
members.
Group I introns require an external G
cofactor for splicing
• First characterized in Tetrahymena.
• Widely distributed in the mitochondrial,
chloroplast, and nuclear genomes of diverse
eukaryotes.
• In animals, group I introns have only been
found so far in the mitochondrial genomes of
one sea anemone and a coral.
Two-step splicing reaction, involving two
transesterification reactions
1. Attack by an external guanine on the 5′ splice
site, adding the G to the 5′ end of the intron
and releasing the first exon.
2. The first exon attacks the 3′ splice site,
ligating the two exons together and releasing
the linear intron.
• Although many group I introns can selfsplice in vitro, many, if not all, require
proteins in vivo to fold into the
catalytically active structure.
• Proteins required for splicing are either
encoded by the introns themselves or by
other genes of the host organism.
Group I intron structure
• The typical secondary structure is
approximately 10 base-paired helical
elements organized into three stacked
domains.
Group II introns require an internal
bulged A for splicing
• Less common than group I introns.
• Found in the mitochondrial and
chloroplast, genomes of certain protists,
fungi, algae, and plants, and in bacterial
genomes.
First transesterification reaction
•
Attack by the 2′-OH of an internal bulged A on
the 5′ splice site.
•
Release of the first exon.
•
Formation of a lariat structure with a 2′→5′
phosphodiester bond.
Second transesterification reaction
•
The first exon attacks the 3′ splice site.
•
The two exons are ligated together.
•
The lariat intron is released, debranched, and
degraded.
• Although many group II introns can selfsplice in vitro, many, if not all, require
proteins in vivo to fold into the
catalytically active structure.
• Proteins required for splicing are either
encoded by the introns themselves or by
other genes of the host organism.
Group II intron structure
• Conserved tertiary structure.
• Six major domains radiating from a
central wheel.
Mobile group I and group II introns
• Spread efficiently into a homologous position in
an allele that lacks the intron.
• Movement is mediated by highly-specific
homing endonucleases that are typically
encoded by the self-splicing intron.
• Group II introns also encode an open reading
frame with homology to reverse transcriptase.
• Group I introns typically move by a process
termed homing.
• Group II introns typically move by a process
termed retrohoming.
Archael introns are spliced by an
endoribonuclease
• Archaea carry introns in their tRNA, rRNA, and
mRNA that are spliced by an archael-specific
mechanism.
• Cut-and-rejoin mechanism that requires ATP,
an endoribonuclease, and a ligase.
• Bulge-helix-bulge motif at the exon-intron
junction.
Some nuclear tRNA genes
contain an intron
• Presence or absence of an intron defines two
classes of eukaryotic nuclear tRNA genes.
• In humans, only tRNATyr and tRNALeu contain
introns.
Nuclear tRNA splicing involves two main
reactions:
1. Cleavage: The intron-containing pre-tRNAs
are cleaved by an endoribonuclease at the 5′
and 3′ boundaries of the intron.
2. Joining: The paired tRNA halves are joined by
ligase.
• In plants and fungi, the 2′ phosphate
and 3′-OH at the splice junction are
resolved by phosphotransferase.
13.4 Cotranscriptional processing
of nuclear pre-mRNA
Eukaryotic mRNA is covalently processed
in three ways prior to export from the
nucleus:
1. Transcripts are capped at their 5′ end with a
methylated guanosine nucleotide.
2. Introns are removed by splicing.
3. 3′ ends are cleaved and extended with a
poly(A) tail.
Cotranscriptional processing of nuclear
pre-mRNA
• The C-terminal domain (CTD) of RNA
polymerase II functions as a “landing
pad” for RNA processing factors.
• Deletion of the RNA polymerase II CTD
inhibits capping, splicing, and poly(A)
cleavage.
•
Experiments have shown that different
regions of the CTD serve distinct functions in
pre-mRNA processing.
•
The C-terminal heptapeptide repeats support
capping, splicing, and 3′ processing.
•
The amino-terminal repeats only support
capping.
Addition of the
5′-7-methylguanosine cap
•
The cap protects mRNA from degradation and
enhances the efficiency of splicing, nuclear
export, and translation.
•
A distinguishing chemical feature of the cap is
the 5′→5′ linkage of 7-methylguanosine to the
initial nucleotide of the mRNA.
•
Often abbreviated as m7GpppN to reflect this
linkage.
The cap is added in three main steps:
•
An RNA triphosphatase removes the terminal
phosphate from the pre-mRNA.
•
Guanylyltransferase adds the capping GMP
from GTP.
•
Methyltransferases methylate the N-7 of the
capping guanosine and the 2′-O-methyl group
of the penultimate nucleotide.
Termination and polyadenylation
•
No consensus termination sequence has
been identified.
•
Most metazoan mRNA 3′ ends are produced
by cleavage of the pre-mRNA at the
polyadenylation site between conserved
AAUAAA and G/U-rich sequence elements.
•
These regions are recognized by cleavage
and polyadenylation specificity factor (CPSF)
and cleavage stimulation factor (CstF).
•
Cleavage requires two additional complexes,
mammalian cleavage factor I and II, CFIm,
CFIIm.
•
The mRNA is cleaved while it is still being
synthesized.
•
Cleavage also occurs at a cotranscriptional
cleavage site (CoTC) which, at least in the
case of the -globin mRNA, is self-cleaving.
•
The remaining transcript still associated with
RNA polymerase II is attacked by the Xrn2
exonuclease that chases after the
polymerase.
•
When the exonuclease catches up,
transcription is terminated.
• The catalytic CoTC core of the -globin
pre-mRNA folds into a defined
secondary structure.
• The minimal catalytic core was shown to
self-cleave in a time course conducted
under protein-free conditions.
•
After cleavage and release of the mRNA, the
transcript is polyadenylated at the 3′ end.
•
Most eukaryotic mRNAs have a chain of A
residues about 100 to 250 nt long.
•
Histone mRNAs have a conserved stem-loop
structure instead of a poly(A) tail.
• The poly(A) tail is added by poly(A)
polymerase.
• Enhances mRNA stability and
translation efficiency.
•
The poly(A) tails are coated with sequencespecific poly(A)-binding proteins.
•
In the nucleus, PABPN1 increases the
processivity of poly(A) polymerase.
•
In the cytoplasm, PABPC functions in the
initiation of translation and the regulation of
mRNA decay.
Oculopharyngeal muscular dystrophy:
trinucleotide repeat expansion in a poly(A)binding protein gene
•
Muscular dystrophy that begins in the eyes
and throat.
•
Prevalence is highest in French-Canadians
who are descended from French immigrants,
a man and wife, who emigrated to Canada in
1634.
•
Trinucleotide repeat expansion in exon 1 of
the PABPN1 gene.
•
Normal protein expanded from 10 alanines in
a row to 12-17 alanines.
•
Leads to intranuclear protein aggregation.
Splicing
• Basic mechanisms is the same as selfsplicing group II introns.
• Except, the 3-D structure required for
splicing is generated by the
spliceosome.
Components of the spliceosome
•
Five uracil-rich small nuclear RNA (snRNA)protein complexes termed snRNPs.
U1, U2, U4, U5, U6
What happened to U3?
•
>200 proteins.
The structure of snRNPs
•
snRNAs are associated with Sm or Sm-like
common core proteins and particle-specific
proteins.
•
Sm proteins are named after the patient
whose autoimmune serum was first used to
detect them.
•
Sm proteins form a doughnut-shaped
complex.
Spinal muscular atrophy: defects in
snRNP biogenesis
•
Fatal autosomal recessive diseases.
•
Degeneration of motor neurons in the anterior
horn of the spinal cord.
•
Mutations in the Survival of Motor Neurons 1
(SMN) gene.
•
Defects in snRNP biogenesis.
SMN-mediated snRNP assembly
in the cytoplasm
•
Assembly of snRNPs is a stepwise process
that takes place in multiple subcellular
compartments.
•
SMN mediates snRNP assembly in the
cytoplasm.
•
SMN forms a pre-import complex with
snRNPs, importin-1 and the import adaptor
snurportin.
SMN is associated with Cajal bodies
and gems in the nucleus
•
The snRNPs enter the nucleus and are
targeted to Cajal bodies.
•
Cajal bodies have numerous roles in the
assembly and/or modification of the nuclear
transcription and RNA processing machinery.
•
Splicing takes place at other locations.
Why do defects in SMN lead to spinal
muscular atrophy?
• SMN is normally expressed at
particularly high levels in motor neurons.
• Neuronal differentiation and
maintenance may have particularly
stringent demands for splicing.
Assembly of the splicing machinery
•
The splicing machinery is recruited to introncontaining transcripts co-transcriptionally.
•
The cap-binding complex is important for this
recruitment.
•
The “splicing cycle” involves the stepwise
assembly of the spliceosome through a series
of short-lived intermediate subcomplexes, at
least in vitro.
•
These subcomplexes can be distinguished by
their different mobilities in native gels or
density gradients, their snRNP composition,
and the stage of processing of the pre-mRNA.
•
An affinity purification method was used to
show that U1 is the first snRNP to bind to premRNA in a yeast nuclear extract.
•
An alternative “penta-snRNP” model has been
proposed based on in vivo analysis.
•
In this model, intron removal is mediated by
pre-existing complexes.
•
Another more recent in vivo analysis is more
consistent with stepwise recruitment of
individual snRNPs, rather than a preformed
penta-snRNP.
Splicing pathway
• Processing events involving RNA
cofactors use the RNA to provide
specificity by complementary base
pairing.
Splicing pathway
1. Formation of the E (early) or
“commitment” complex
•
•
•
U1 snRNP binds to the conserved 5′ GU
splice site.
The 65 kD subunit of U2 auxiliary factor
(U2AF) binds to the polypyrimidine tract.
The 35 kDa subunit of U2AF binds to the 3′
AG splice site.
2. Formation of the A complex or “prespliceosome”
•
U2 snRNP binds to the branch site region.
3. Formation of the B complex
•
The A complex is joined by the U4/U6/U5 trisnRNP.
4. Formation of the C complex or
“spliceosome”
•
•
•
•
Rearrangement of the B complex.
U1 and U4 snRNPs are lost from the complex.
U6 snRNP base pairs at the 5′ splice site and
with U2 snRNP.
U5 snRNP interacts with sequences in the 5 ′
and 3′ exon.
5. Splicing proceeds by two
transesterification reactions
•
•
•
•
Lariat-shaped intermediate as in group II
intron splicing.
Ligation of the two exons and release of the
intron.
Spliceosome is disassembled.
The lariat intron is debranched and degraded.
Protein factors that help
mediate splicing
• Dynamic remodeling of the spliceosome
requires DEXH/D box RNA helicases.
• SC35 and ASF/SF2, members of the
highly conserved serine/arginine (SR)rich family of splicing factors have a dual
role in stimulating constitutive and
regulated splicing.
•
SR proteins have a modular domain structure:
– One or two N-terminal RNA recognition
motifs (RRMs) for sequence-specific RNA
binding.
– C-terminal “RS domain”: repeated arginineserine dipeptides that can be
phosphorylated at multiple positions.
•
In the interphase nucleus, the bulk of SR
proteins are located in “speckles.”
A model for the regulation of splicing
• The recruitment of splicing factors is
controlled by activating and inhibitory
splice regulatory proteins.
• The splice regulatory proteins bind
exonic and intronic splicing enhancers
and silencers.
The U12-type intron splicing pathway
•
A minor class of introns (0.15 to 0.34%).
•
Variant but highly conserved 5′ splice sites
and branch points .
•
Spliced with the help of variant classes of
snRNAs, including U11, U12, U4atac, and
U6atac.
Is the spliceosome a ribozyme?
•
Only a few components of the spliceosome
interact directly with the pre-mRNA substrate:
protein Prp8, U2 snRNA, and U6 snRNA.
•
Prp8, a component of the U5 snRNP, is
proposed to function as a cofactor in RNAmediated catalysis.
•
There is strong experimental evidence that U2
and U6 snRNA contribute to the catalysis of
pre-mRNA splicing.
Experimental Example
•
Formation of “RNA X”, a product similar to the
first step of splicing, is mediated by U2 and
U6 snRNA in vitro.
Prp8 gene mutations cause
retinitis pigmentosum
•
Genetic disorder causing degeneration of
photoreceptors in the retina.
•
Severe autosomal dominant form associated
with mutations in the gene encoding Prp8, an
essential component of the spliceosome.
•
Rod photoreceptor cells may be particularly
sensitive to alterations in the splicing
machinery because of their rapid turnover.
13.5 Alternative splicing
• A versatile means of regulating gene
expression.
• Mechanism for generating protein
diversity from a small set of genes.
Alternative splicing
•
A typical human or mouse gene contains 8-10
exons which can be joined in different
arrangements by alternative splicing.
•
Splicing of most exons is constitutive.
•
Splicing of some exons is regulated.
•
At least 74% of human genes are alternatively
spliced.
Effects of alternative splicing on
gene expression
•
Multiple alternative splicing positions in premRNAs gives rise to a family of related
proteins.
•
Alternative splicing of 5′ or 3′ untranslated
regions affects elements that regulate
translation, stability, or mRNA localization.
•
Insertion of premature termination codons
leads to nonsense-mediated decay.
The DSCAM gene:
extreme alternative splicing
•
•
•
•
Drosophila gene encoding the Downs
syndrome cell adhesion molecule (Dscam), a
transmembrane neuronal cell adhesion
molecule.
96 variable exons out of a total of 115.
38,016 potential different protein isoforms.
Human DSCAM gene has 30 exons and only
three alternatively spliced transcripts.
Regulation of alternative splicing
•
Cis-acting regulatory sequences are bound by
trans-acting proteins that regulate splicing.
•
Exonic or intronic splicing enhancers.
•
Exonic or intronic splicing silencers.
•
Histone modifications may affect splicing
outcome.
Alternative splicing in mammalian
heart development
Alternative splicing of Ca2+/calmodulin-dependent
kinase II  (CaMKII )
•
•
•
Neuronal isoform (A) is targeted to the
transverse tubules of the sarcolemmal
membranes.
Cardiac isoform (B) is targeted to the
nucleus.
Cardiac isoform (C) is located in the
cytoplasm of muscle cells.
•
In the developing fetal heart, all three
isoforms are expressed.
•
Between 1-2 months, only the cardiac
isoforms are expressed.
•
Inappropriate expression of the neuronal
isoform (A) leads to defects in heart
development and function.
Trans-splicing
•
The exon from one pre-mRNA joins to an
exon from another pre-RNA.
•
A rare event.
•
Occurs in special situations in organisms as
diverse as flatworms, the protist Euglena
gracilis, plant organelles, nematodes, and
Drosophila.
Trans-splicing
Three major types
• Discontinuous group II trans-splicing
• Spliced leader trans-splicing
• tRNA trans-splicing
Discontinuous group II trans-splicing
•
First discovered in plant chloroplast genomes.
•
Coding sequences are joined from separate
transcripts to form a complete opening
reading frame.
•
Example: Trans-splicing events in NADH
dehydrogenase 1 (nad1) pre-mRNA
Spliced leader trans-splicing
•
First discovered in African trypanosomes.
•
A short sequence is added to the 5′ termini of
mRNAs.
•
The leader exon functions to:
–
–
–
provide a 5′ cap structure.
resolve polycistronic transcripts into individual
capped RNAs.
enhance mRNA translational efficiency.
tRNA trans-splicing
• In Nanoarchaeum equitans, nine genes
encode separate tRNA halves which are
then joined by trans-splicing.
13.6 RNA editing
•
Post-transcriptional modification of the base
sequence of mRNA.
•
Widespread mechanism for changing genespecified codons and thus protein structure
and function.
•
First discovered in trypanosomes, then found
in viruses, plants, slime molds, humans,
marsupials, squid, dinoflagellates, etc.
RNA editing in trypanosomes
•
Early 1980s: Routine study of mitochondrial
DNA of African trypanosomes.
•
Major RNA transcripts encoding important
electron transport chain enzymes found that
contained nucleotides not encoded in the
DNA.
•
Was “The Central Dogma” in jeopardy?
•
Extensive post-transcriptional editing (“panediting”) of the cytochrome oxidase III
transcript in Trypanosoma brucei.
•
Cytochrome-b mRNA edited in procyclic-form
parasites in the nutrient-poor environment of
the tsetse fly, and unedited in bloodstream
forms within mammalian hosts.
Editing events: uridine (U) insertions or
deletions
•
Correct internal frameshifts.
•
Create AUG start codons and UAG/UAA stop
codons.
•
Create open reading frames.
Guide RNAs
1990: The editing mechanism was
discovered
•
U-insertions and deletions are mediated by
multiple short guide RNAs (gRNAs) that
specify the edited sequence.
•
Catalyzed by a multiprotein complex called
the editosome.
• Pre-edited mRNAs are encoded by the
larger maxicircles of the mitochondrial
DNA genome (kinetoplast).
• Guide RNAs are encoded by the smaller
minicircles.
Mechanism of editing
•
Mechanism determined by in vitro editing
assays, mainly in mitochondrial extracts from
procyclic-form trypanosomes.
•
Recent development of an in vitro system
from mammalian bloodstream-form
trypanosomes recreates complete cycles of
both insertion and deletion.
•
Multiple gRNAs are required to edit each premRNA transcript.
•
gRNAs are precise complementary versions
of mature mRNAs in the edited region.
•
The gRNA provides specificity by
complementary base pairing (including GU
base pairs).
•
Editing occurs from 3′→5′ along the mRNA.
A series of enzymatic reactions catalyzed
by the editosome
1. Anchoring: Anchor duplex forms between the
pre-mRNA and the gRNA by complementary
base pairing.
2. Cleavage: Cleavage at the first site of
mismatch by an endoribonuclease.
3. Uridine insertion or deletion: A U is inserted
by a 3′ terminal uridylyl transferase (TUTase)
or removed by a U-specific endoribonuclease
(ExoUase).
4. Ligation: RNA ends are joined by RNA ligase
5. Repeat of editing cycle: The cycle is repeated
until the RNA is fully edited.
RNA editing in mammals
•
Two main classes of editing enzymes that
deaminate encoded nucleotides.
•
Adenosine to inosine (A→I) editing.
•
Cytidine to uridine (C→U) editing.
•
Regulate a diversity of processes, including
aspects of neurotransmission and lipid
metabolism.
Adenosine to inosine
(A→I) editing
•
Catalyzed by double-stranded RNA specific
ADAR (adenosine deaminase acting on RNA)
family.
•
Affects >1600 genes: most editing events are
within introns and Alu elements in UTRs.
•
Altered editing patterns associated with
inflammation, epilepsy, depression, malignant
gliomas, and amyotrophic lateral sclerosis.
Structure of ADAR
• Crystal structure revealed an unusual
feature.
• The enzyme requires inositol
hexakisphosphate as a cofactor.
Amyotrophic lateral sclerosis:
a defect in RNA editing?
•
“Lou Gehrig’s disease.”
•
Selective loss of upper and lower motor
neurons starting in mid-life.
•
Muscle wasting and progressive paralysis.
•
There is no cure.
Familial amyotrotrophic lateral sclerosis
(ALS)
•
Linked to defects in genes encoding
superoxide dismutase 1 (SOD1) and
senataxin
Sporadic (nonhereditary) (ALS)
•
•
Accounts for most cases of the disease.
Cause remains unknown.
Hypothesis for cause of sporadic ALS
•
Defect in RNA editing of AMPA receptors (a
subtype of glutamate receptors) due to a
reduction in ADAR2 deaminase activity.
•
AMPA receptors are composed of four
subunits in various combinations.
•
Almost all GluR2 mRNA undergoes A→I
editing.
•
Editing changes glutamate (Q) codon to
arginine (R) codon.
•
The Ca2+ conductance of AMPA receptors
varies depending on whether the edited
GluR2 subunit is a component of the receptor.
•
AMPA receptors lacking GluR2 or containing
unedited GluR2 (GluR2Q) are Ca2+
permeable.
•
This leads to excitotoxicity and neuronal
death.
Cytidine to uridine (C→U) editing
• Only identified in two gene transcripts in
mammals
– Apolipoprotein B editing
– Neurofibromatosis type 1 editing
Apolipoprotein B editing
in humans
•
ApoB is a plasma protein that plays a key role
in the assembly, transport, and metabolism of
plasma lipoproteins.
ApoB-100: liver specific
ApoB-48: small intestine
•
The two different proteins are products of a
single gene generated by RNA editing.
•
The apoB gene has 29 exons and 28 introns.
•
Within the nucleus, the pre-mRNA undergoes
splicing, polyadenylation, and editing in cells
of the small intestine.
•
The C→U transition converts a glutamine
codon (CAA) to a stop codon (UAA).
•
There are 375 CAA triplets in the ApoB gene,
of which 100 are in-frame glutamine codons.
•
Even so, the editing complex is able to confer
almost absolute specificity at the correct CAA.
•
Binding of ACF to the RNA transcript positions
APOBEC1 over the correct cytidine.
• Development of an in vitro editing assay
lead to significant advances in
understanding the molecular
mechanisms for C→U editing.
13.7 Post-transcriptional gene
regulation by RNAi
• The recent discovery of hundreds of
genes that encode small regulatory RNA
molecules represents another landmark
discovery in molecular biology.
• Why were small regulatory RNAs
overlooked until just recently?
• RNA interference (RNAi) is a sequencespecific gene-silencing process that
occurs at the post-transcriptional level.
• In 2002, the journal Science designated
RNAi as the “breakthrough of the year.”
Two major classes of small regulatory
RNAs
• MicroRNA (miRNA)
• Small interfering RNA (siRNA)
MicroRNA (miRNA)
•
Post-transcriptional gene regulation.
•
Hetero-silencing: derived from unique genes
that specify the silencing of different genes.
•
First discovered in the early 1990s in the
nematode worm C. elegans.
•
Now, hundreds of different human miRNAs
have been identified.
Small interfering RNA (siRNA)
•
Defense of the genome.
•
Auto-silencing: guide the silencing of the
same genetic locus or a very similar locus
from which they originate:
– Viruses.
– Transposable elements.
– Heterochromatin.
– dsRNA inserted into a cell by the
bench scientist.
RNAi is triggered by double-stranded RNA
(dsRNA) molecules.
1. Dicer, a specialized RNase III family
ribonuclease processes dsRNA into short
siRNAs of ~21-26 nt in length with twonucleotide 3′-overhangs.
2. The siRNAs trigger formation of an RNAinduced silencing complex (RISC).
3. The ATP-dependent unwinding of the siRNA
duplex by helicase activity in the RISC loading
complex leads to activated RISC.
4. The single-stranded siRNA is used as a guide
for target RNA recognition (viral or cellular
RNA) and cleavage by Slicer activity.
5. In worms, flies, plants, and fungi, RNAdirected RNA polymerase (RdRP) uses the
siRNA antisense strands as primers and
makes new dsRNA.
• Many bacteria and archaea also protect
themselves from viruses through genetic
interference pathways.
• In this case, the small RNAs are encoded by
clustered, regularly spaced palindromic repeats,
called CRISPR RNAs.
• CRISPR RNAs specify the cleavage of foreign
DNA.
The discovery of RNAi
Before RNAi was well-characterized the
process was referred to by a number of
names:
• Post-transcriptional gene silencing (plants).
• Quelling (fungi)
• RNAi (animals)
Experiments by Andrew Fire, Craig Mello
and colleagues in 1998
• Discovered that injecting dsRNA into the body
cavity of Caenorhabditis elegans worms
silenced only mRNAs containing a
complementary sequence.
• Fire and Mello were awarded the Noble Prize
in 2006 for their discovery of RNAi.
RNAi machinery
• Gene silencing is carried out by RISC, the
RNA-induced silencing complex.
• Experiments have shown that the Argonaute
subunit of RISC has “Slicer” activity.
– PAZ (Piwi Argonaute Zwille) domain: RNA binding.
– PIWI domain: nuclease.
The discovery of miRNA in
Caenorhabditis elegans
Heterochronic gene hierarchy
•
Lin-4 miRNA regulates the timing of larval
development in C. elegans.
•
Lin-4 miRNA base pairs with the 3′ UTR of lin14 mRNA and lin-28 mRNA.
•
Originally thought to block mRNA translation.
• More recent results suggest that lin-4
miRNA also promotes degradation of
lin-14 and lin-28 mRNA.
• When let-7 miRNA was discovered 7
years after lin-4, it became clear that
this type of regulatory mechanism was
not unique.
• Discovery of RNAi and siRNAs
dramatically increased interest in small
regulatory RNAs.
Processing of miRNAs
• miRNAs are processed from gene
transcripts that form hairpin structures.
• Many human miRNA genes (~56%) are
located within the introns of proteincoding pre-mRNAs.
miRNAs biogenesis and function involves
six main steps
1. Transcription: The miRNA gene is
transcribed, possibly by RNA polymerase II.
2. Cleavage by Drosha: The primary miRNA (primiRNA) hairpin is processed by Drosha in the
nucleus.
3. Nuclear export: The pre-miRNA exits the
nucleus by an exportin 5-mediated pathway.
4. Cleavage by Dicer: The pre-miRNA is cleaved
into duplex miRNA by Dicer.
5. RISC formation: The duplex is unwound by a
helicase in the RISC loading complex and the
mature single-stranded miRNA associates
with activated RISC.
6. mRNA inhibition: RISC targets mRNAs for
either degradation or translational repression
depending on the degree of complementarity.
miRNAs target mRNA for degradation
and translational inhibition
•
Short complementary regions in 3′-UTR:
– Translational repression
•
Extensive complementarity in coding regions
or UTR:
– mRNA degradation
•
100s of miRNAs have been cloned or predicted
•
Play a role in:
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•
Apoptosis.
Neuronal asymmetry and brain morphogenesis.
Leaf and flower development.
Development of cancer.
Fortuitous anti-viral defense.
Tissue specific regulation of mRNA levels.
The function of the majority remains unclear.
Tissue-specific gene expression for
genes downregulated by miRNAs
•
miRNAs were transfected into human cells
and microarray analysis was used to examine
changes in mRNA expression profiles.
•
Delivery of muscle-specific miR-1 shifted the
profile towards that of muscle.
•
Delivery of brain-specific miR-124 shifted the
profile towards that of brain.
Antiviral defense
• Cellular miRNA was shown to restrict
accumulation of primate foamy virus
type 1 in human cells.
• Fortuitous recognition of a
complementary sequence in the viral
RNA by miRNA-32.
13.8 RNA turnover in the nucleus
and cytoplasm
•
Quality control pathways in the nucleus.
•
Some mRNAs are stored in the cytoplasm.
•
Most mRNAs are immediately translated and
later degraded.
•
Nonsense-mediated decay of mRNA.
Nuclear exosomes and quality control
•
Short-lived processing intermediates of
rRNAs, snoRNAs, and snRNAs are degraded
by the nuclear exosome from 3′→5′.
•
Specific nuclear decay pathways destroy
defective pre-mRNAs or those failing to form
export-competent mRNPs.
•
The TRAMP complex adds a short poly(A) tail
to target molecules before recruitment and
activation of the exosome.
•
TREX and SR proteins recruit general nuclear
export factors, such as TAP/NFX1 to mRNAs.
•
Contribute to the ability of the export
machinery to discriminate between spliced
and unspliced mRNPs.
•
Further quality control surveillance and
retention of unspliced transcripts occurs at the
nuclear pore complex.
Cytoplasmic RNA turnover
• Once in the cytoplasm there are several
possible fates for an mRNA:
– Held in a translationally silent state.
– Translated and then degraded.
– Nonsense-mediated mRNA decay.
Storage of translationally silent mRNA
• Mediated by deadenylation at a
particular stage of development.
• Re-addition of the poly(A) tail later in
development.
General mRNA decay pathways
•
Most mRNAs are immediately translated and
later degraded by general decay pathways.
•
The 5′ cap is removed by decapping
enzymes, followed by exonuclease digestion
from 5′→3′.
•
Alternatively, the 3′ poly(A) tail is removed by
deadenylases and the rest of the mRNA is
degraded by exonucleases from 3′→5′.
Nonsense-mediated
mRNA decay
•
mRNAs that contain a premature termination
codon are rapidly degraded by nonsensemediated mRNA decay.
•
Exon junction complexes (EJCs) are
assembled near exon-exon boundaries after
mRNA splicing.
•
When the ribosome begins translating an
mRNA, the EJCs are normally displaced.
•
If a premature termination codon is present,
this activates a surveillance complex.
•
The surveillance complex interacts with the
prematurely terminating ribosome and targets
the mRNA for degradation.