Topic 27

Post Transcriptional Regulation

This Slide Property of Jeff Singer

Concepts – Post-transcriptional regulation
• Transcriptional Attenuation and
Riboswitches
• Alternative splicing
• mRNA: Localization and processing
• RNA editing
• non-coding RNAs
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Outline Post-transcriptional regulation

1. mRNA processing 2. Translational contro
control 3. short non-coding
a. Attenuation and RNA
Riboswitche 4. long non-coding
b. Alternative splicin RNAs (lncRNA)
c. RNA editin

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 gene expression to daughter cells.

Posttranscriptional regulation pathways START RNA

POSITIVE protein A CONFORMATION normal folded
FEEDBACK CHANGE TO protein TRANSCRIPTION
not made
LOOP AGGREGATED
protein A made STATE POSSIBLE RNA
ACTIVATED misfolded protein (prion)
1. Attenuation
A NEW GENE NEW PROTEIN
ATTENUATION
CAPPING
transcript
aborts

EXPRESSION CONFORMATION nonfunctional

SPLICING
2. Alternative splicing
A
PATTERN
INHERITED
STATE
INHERITED AND 3′-END
mRNA
sequences
A

A
CLEAVAGE
POSSIBLE

3. mRNA cleavage
POSITIVE FEEDBACK LOOP BY
TRANSCRIPTION REGULATOR
PROTEIN AGGREGATION STATE
RNA EDITING

NUCLEAR retention and

EXPORT degradation
(B) EPIGENETIC MECHANISMS THAT ACT IN TRANS
4. RNA Editing in nucleus

Summary SPATIAL

5. cellsNuclear
Eukaryotic can use inheritedtransport
forms of DNA methylation and inherited states
LOCALIZATION
IN CYTOPLASM
of chromatin condensation as additional mechanisms for generating cell memory
START
6. Localization
of gene expression patterns. An especially dramatic case that involves chromatin
condensation is the inactivation of an entire X chromosome in female mammals.
TRANSLATION
translation
blocked

DNA methylation underlies the phenomenon in mammals of genomic imprinting, POSSIBLE

7. Translational control
in which the expression of a gene depends on whether it was inherited from the
mother or the father.
TRANSLATIONAL
RECODING
MBoC6 m7.445/7.54 POSSIBLE

8. mRNA stability
POST-TRANSCRIPTIONAL CONTROLS
RNA
STABILIZATION
RNA degraded

CONTINUED
In principle, every step required for the process of gene expression can be con- PROTEIN SYNTHESIS
trolled. Indeed, one can find examples of each type of regulation, and many genes
are regulated by multiple mechanisms. As we have seen, controls on the initiation Figure 7–54 Post-transcriptional
This Slide Property of Jeff Singer

Attenuation and the Trp operon

The control region of the trp operon codes

for a leader peptide.
Translation can control transcription
termination.

This Slide Property of Jeff Singer

Attenuation and the TRP operon

Alternative secondary structures control

termination. Tryptophan controls ribosome
position.

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 Riboswitch

A riboswitch is a regulatory segment of a messenger RNA molecule

that binds a small molecule, resulting in a change in production of the
proteins encoded by the mRNA

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 Attenuation and Riboswitches
(A)
genes for purine
biosynthesis ON (B)
genes for purine
biosynthesis OFF

Transcription Attenuation Causes the Premature Termination

• Changes transcription termination hairpin loop dependent on Guanine of
Some RNA Molecules
concentratio
It has long been known that the expression of some genes is inhibited by pre-
• 414
Other examples Control ribosome
Chapter 7: cause of Gene Expression
stalling atophenomenon
prevent the hairpin fromattenua-
mature termination of transcription, called transcription G
forming tion. In some of these cases, the nascent RNA chain adopts a structure that causes
it to interact with the RNA polymerase in such a way as to abort its transcription.
riboswitch
When the gene product is required, regulatory proteins bind to the nascent RNA
chain and remove the attenuation, allowing the transcription ofguanine
a complete RNA
molecule. (C)
A well-studied example of transcription attenuation occurs during G the life
cycle of HIV, the human immunodeficiency virus that is the causative agent of
acquired immune deficiency syndrome, or AIDS. Once the HIV genome has been transcription
Figure 7–55 A riboswitch that responds
integrated into the host genome, the viral DNA is transcribed by the cell’s RNA toterminator
guanine. (A) In this example from
polymerase II (see Figure 5–62). However, this polymerase usually terminates bacteria, the riboswitch controls expression
RNA synthesizing
polymerase transcripts of several hundred nucleotides and of the purine biosynthetic genes. When
transcription after guanine levels in cells are low, an
therefore fails to efficiently transcribe the entire viral genome. When conditions elongating RNA polymerase transcribes
for viral growth are optimal, a virus-encoded protein called Tat, which binds to the purine biosynthetic genes, and the
a specific stem-loop structure in the nascent RNA that contains a “bulged base,” enzymes needed for guanine synthesis are
therefore expressed.
genes (B)
forWhen guanine is
prevents this premature termination
genes for purine(see Figure 6–89). Once bound to this spe- purine
(A) biosynthesis (B) abundant, it binds the riboswitch,
OFF causing
cific RNA structure (called TAR), ON
Tat assembles several host-cell proteins that biosynthesis
it to undergo a conformational change that
MBoC6 m7.93/7.56
allow the RNA polymerase to continue transcribing. The normal role of at least forces the RNA polymerase to terminate

Transcription Attenuation Causes Bacteria or Eukaryotes?

some of these proteins is to prevent pausing and premature termination by RNA
the Premature Termination of transcription (see Figure 6–11). (C) Guanine
(red) bound to the riboswitch. Only those
polymerase when it transcribes normal cell genes. Thus, a normal cell mechanism
Some RNA Molecules has apparently been highjacked by HIV to permit transcription of its genome to be nucleotides that form the guanine-binding
pocketSlide
are shown. Many other riboswitches
controlled by a single viral protein. This Property of Jeff Singer
n

 Prokaryotic Riboswitches

Cell 152, January 17, 2013

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 Eukaryotic Riboswitches
thiamine
pyrophosphat
(TPP)

Cell 152, January 17, 2013

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a single gene can produce dozens of different proteins. In one extreme case, a MBoC6 m7.94/7.57

Alternative splicing
Drosophila gene may produce as many as 38,000 different proteins from a single
gene through alternative splicing (Figure 7–57), although only a fraction of these
forms have thus far been experimentally observed. Considering that the Drosoph-
Drosophila genome predicts 14,000 genes!
ila genome has approximately 14,000 identified genes, it is clear that the protein
complexity of an organism can greatly exceed the number of its genes. This exam-
ple also illustrates the perils in equating gene number with an organism’s com-
This gene alone can create 38,000!
plexity. For example, alternative splicing is rare in single-celled budding yeasts

A exons B exons C exons D exons

1 12 1 48 1 33 12

Dscam gene

A8 C16

mRNA

B24 D2
one out of 38,016 possible splicing patterns

Figure 7–57 Alternative splicing of RNA transcripts of the Drosophila Dscam gene. DSCAM proteins have several different functions. In cells
This Slide Property of Jeff Singer

POST-TRANSCRIPTIONAL CONTROLS Alternative splicing 415

1.Exon skipping
5 patterns: Figure 7–56 Five patterns of alternative RNA splicing. In each case, a
single type of RNA transcript is spliced in two alternative ways to produce
two distinct mRNAs (1 and 2). The dark blue boxes mark exon sequences
exon skipping

1
that are retained in both mRNAs. The light blue boxes mark possible exon 2
sequences that are included in only one of the mRNAs. The boxes are
2.Intron retention
joined by red lines to indicate where intron sequences (yellow) are removed.
(Adapted from H. Keren et al. Nat. Rev. Genet. 11:345–355, 2010. With
intron retention

permission from Macmillan Publishers Ltd.)

1
2
Riboswitches are perhaps the most economical examples of gene control
3.Alterative 5’ splice site
devices, inasmuch as they bypass the need for regulatory proteins altogether. In alternative 5′ splice site
the example shown in Figure 7–55, the riboswitch controls transcription elonga-
1
tion, but they can also regulate other steps in gene expression, as we shall see later 2
in this chapter. Clearly, highly sophisticated gene control devices can be made
from short sequences of RNA, a fact that supports the hypothesis of an early “RNA
world.” 4.Alternative 3’ splice site alternative 3′ splice site

1
2
Alternative RNA Splicing Can Produce Different Forms of a Protein
from the Same Gene
5.Mutually exclusive exons:
As discussed in Chapter 6 (see Figure 6–26), RNA splicing shortens the transcripts
mutually exclusive exons

of many eukaryotic genes by removing the intron sequences from the mRNA pre- 1
2
cursor. We also saw that a cell can splice an RNA transcript differently and thereby
make different polypeptide chains from the same gene—a process called alterna-
tive RNA splicing (Figure 7–56). A substantial proportion of animal genes (esti- This Slide Property of Jeff Singer
:

 Alternative splicing: Regulation

Alternative splicing can be passive
1.Due to sequence ambiguity, several possible splicing
events can occur at random

2.Many cases involve regulation and result in non-random

splicing, this regulation can be positive or negative

This Slide Property of Jeff Singer

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 Alternative Splicing
Alternative splicing is regulated
1. 5% of genes do this in yeast, 75% in human
2. Negative Regulation Mechanis
• Blocking splice sit

• “Strong” blocked, “weak” use

3.Positive Regulation Mechanis

• Stimulating use of “weak” splice site
This Slide Property of Jeff Singer
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sequences can be assembled in more than one way raised new questions about
the definition of a gene. A gene was first clearly defined in molecular terms in
Alternative splicing: Regulation
the early 1940s from work on the biochemical genetics of the fungus Neurospora.
repressor
Figure 7–5
control of a
R (A) In negat
pre-mRNA transcript
(A) NEGATIVE binds to a s
CONTROL mRNA trans
the splicing
SPLICING NO SPLICING
This often re
splice site, t
mRNA mRNA pattern of s
(B) In positiv
machinery i
intron sequ
activator assistance
pre-mRNA transcript A Because RN
(B) POSITIVE sequences
CONTROL be located
SPLICING the splice ju
NO SPLICING
are often ca
analogy wit
mRNA mRNA mentioned

This Slide Property of Jeff Singer

 Fly sex determination

Fly sex determination pathway is regulated

by alternative splicing
• Sxl is a splicing represso
• Tra is a splicing activato
• Dsx is a transcription repressor

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:

Fly sex determination

X:A ratio sensing mechanism turns
on SXL expression in females

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Alternative mRNA cleavage in immunoglobulins
• CstF-stimulates usage of a sub-optimal 418
poly AChapter
site that leads of
7: Control toGene
a shorter transcript and a secreted
Expression
form of the antibody
• When activated to secrete, CstF is upregulated polyadenylation sites polyadenylation sites
weak strong weak strong

5′ 5′
DNA 3′ DNA 3′

CstF
CstF
pre-mRNA AAA pre-mRNA AAA

3′ noncoding RNA 3′ noncoding RNA

CstF-Cleavage stimulatory mRNA AAA mRNA AAA

factor-induced when cells

enter S-phase
B cell with membrane- B cell secretes antibodies
bound antibodies
(A) resting B cell, low levels of CstF (B) activated B cell, high levels of CstF

CstF low CstF high

of the longer RNA transcript. When activated to produce antibodies, the B lym-
phocyte increases its CstF concentration; as a result, cleavage now occurs at the
suboptimal site, and the shorterMBoC6
transcript isThis
m7.99/7.60 Slide Property
produced. of Jeff
In this way, Singerin
a change

RNA editing-Addition of U nucleotides

•Guide RNAs (encoded by the
genome) direct sequence changes
in the original message that add
and remove uridine nucleotides
that alter the original reading
frame and sequence

•Utilize an anchor sequence and

U nucleotides are added by an
enzyme called uridylyl
transferas

•Seen in the mitochondria of

trypanosomes

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NA editing occur: the deamination of

RNA editing-A to I and C to U editing
and, less frequently, the deamination
ng), as shown in Figure 5–43. Because
ng properties
Seen of the bases (I pairs
in mammals-Can with
change
ndprotein
effects on the meaning
coding, of theand
splicing RNA.
hange the
transpor amino acid sequence of the
ADAR
reating a premature stop codon.
o Deamination of Adenine to produce Edits enzyme

A
ffect the pattern
Inosine-ADAR of pre-mRNA splicing,
theo cytosol, the efficiency with which
Deamination of Cytosine to produce
between microRNAs (miRNAs) and
Uracil
t will be discussed later in the chapter.
ularly prevalent in humans, where it exon intron
Might be a defense mechanism 5′ 3′
mes called ADARs (adenosine deam-
against viral infections
f editing; these enzymes recognize a The edit occurs on a sequence that contains duplex RNA
med through base-pairing between the Figure 7–60 Mechanism (a stem) of A-to-I RNA

uence located elsewhere on the same editing in mammals. Typically, a sequence

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 Regulated nuclear transport

HIV genome
①One long transcript makes 30 different mRNA molecule
②Uses many different splicing pattern
420 Chapter 7: Control of Gene Expression
Problem: How to get the mature RNA (unspliced) into the cytoplasm to package?

Vpr Vpu Nef

Vif
Env
Pol
Gag Tat viral DNA
Rev integrated
into host
genome
1,000 nucleotide pairs
5′ splice sites RRE
viral RNA
3′ splice sites
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:

 pro
replicate by providing the gene products in roughly the order in which they are tra
needed (Figure 7–63). Regulation by Rev and by Tat, the HIV protein that counter- int

Regulated nuclear transport acts premature transcription termination (see p. 414), allows the virus to achieve
latency, a condition in which the HIV genome has become integrated into the
host-cell genome but the production of viral proteins has temporarily ceased.
ge
the
the
Tat
pro
(A) early HIV synthesis exa
(se

Early in infection integrated viral DNA

elo
int

Rev is made and is cytoplasmic unspliced

Rev and other early
viral proteins
RNAs synthesized

fully spliced
nuclear mRNAs
retention and
eventual degradation

Late in infection NUCLEUS CYTOSOL

Rev concentration increases, (B) late HIV synthesis

enters nucleus, and binds integrated viral DNA

unspliced RNA targeting it for Fig

by
infe

export all viral

proteins
synthesized
co
an
tra
ha
int
be
the
NUCLEUS CYTOSOL the
ne

This Slide Property of Jeff Singer

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 function.
Several mechanisms for mRNA localization have been discovered (Figur

mRNA localization
7–64), all of which require specific signals in the mRNA itself. These signals ar
usually concentrated in the 3ʹ untranslated region (UTR), the region of RNA tha

Three methods for

cytoplasmic RNA
localization
W/UTR
1.Direct transpor
2.Random diffusion and trappin
3.Degradation and local protection
W/O UTR
by trapping

directed transport random diffusion generalized degradation

on cytoskeleton and trapping in combination with local
protection by trapping

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of Gene Expression mRNA localization

o The signals for localization reside in the 3’ UTR
An experiment demonstrating the importance of the 3ʹ
(untranslated region
zing mRNAs to specific regions of the cytoplasm. For
o This
nt, two region
different typicallylabeled
fluorescently contains
RNAs localization
were prepared by
information
NA in vitro in the presence of fluorescently labeled derivatives of
A (labeled with a red fluorochrome) contains the coding region
phila Hairy protein and includes the adjacent 3ʹ UTR (see Figure
her RNA Here(labeled RNAcontains
green)
the same with thethe Hairy
3’ UTR coding
(red) or region with
leted.without
The twothe RNAs were
3’ UTR mixedare
(green) and injected
made in theinto a Drosophila
tage of development
same animal: when multiple nuclei reside in a common
e Figure 7–26). When the fluorescent RNAs were visualized
er, theThe
full-length hairy RNA
UTR containing (red)was
RNA(red) localized to the apical
correctly
but the transcript
blue)localizes missing
whereas the the 3ʹ the
one missing UTRsignal
(green) failed to
is one(green)
of many transcriptional
does not. regulators that specify positional
the developing Drosophila embryo (discussed in Chapter 21),
ation of its mRNA (shown in this experiment to depend on its
cal for proper fly development. (Courtesy of Simon Bullock and
owicz.)
20 m
This Slide Property of Jeff Singer
µ

 in kinases that respond to the changes in conditions.

Translational control
The normal function of eIF2 was outlined in Chapter 6. It forms a complex
GTP and mediates the binding of the methionyl initiator tRNA to the small
omal subunit, which then binds to the 5ʹ end of the mRNA and begins scan-
Block
along Shine-dalgarno
the mRNA. When an AUG codon oris AUG
recognized, the eIF2 protein hydro-
the bound GTP to GDP,
A.Translationcausing a conformational
repression change
from a in the protein
ribosomal and
protei
sing it from the small ribosomal subunit. The large ribosomal subunit then
B.Temperature
the small one regulates
to form a complete ribosome AUGprotein
that begins access synthesis.

AUG
STOP STOP
AUG
5′ 3′ 5′ 3′ OFF
H2N COOH ON

protein made
INCREASED
TEMPERATURE
translation repressor protein
STOP
AUG
STOP 5′ 3′
AUG OFF COOH ON
5′ 3′ H2N
no protein made
(A) (B)

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 AUG
Translational Control
STOP STOP
AUG
5′ 3′ 5′ 3′ OFF
H2N COOH ON

protein made
INCREASED
Block Shine-dalgarno or AUG TEMPERATURE
translation repressor protein
STOP
C. Binding of aSTOP small molecule to a riboswitch
5′ that
AUG blocks SD
3′
AUG OFF COOH ON
5′ 3′ H N
sequencno protein made 2

(A) (B)
D. An antisense RNA blocks the SD sequence

5′ STOP STOP
AUG AUG
3′ 5′ 3′ ON
H2N COOH ON H 2N COOH

small molecule

STOP
AUG
AUG

STOP 5′ 3′
OFF
3′ OFF
5′ 3′ antisense RNA 5′
(C) (D)

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 with a stop codon, with no stop codons in between—that lie between the 5ʹ end of

Global translational repression

the mRNA and the beginning of the gene. Often, the amino acid sequences coded
by these upstream open reading frames (uORFs) are not important; rather, the
uORFs serve a purely regulatory function. An uORF present on an mRNA mol-
ecule will generally decrease translation of the downstream gene by trapping a

guanine nucleotide exchange

factor, eIF2B

inactive active
eIF2 eIF2
Phosphorylation of eIF-2-can occur
under conditions of stress GDP GDP GTP

• Blocks recyclin GDP

(A)

• Part of the mechanism for the

eIF2B
establishment of G0 (quiescence)
inactive IN ABSENCE OF
elF2 P P ACTIVE eIF2B,
THE REMAINING
GDP GDP GDP UNBOUND eIF2
REMAINS IN ITS
INACTIVE, GDP-
PROTEIN KINASE PHOSPHORYLATED
BOUND FORM
PHOSPHORYLATES eIF2 SEQUESTERS
AND PROTEIN
eIF2 ALL eIF2B AS AN
(B) SYNTHESIS SLOWS
INACTIVE COMPLEX
DRAMATICALLY

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 Translational regulation
Leaky scannin
1.Sometimes first AUG not in a good regio
2.Might go to next AU
3.Can be used to make several versions of the same
protein with different 5’ end
Upstream ORF
1.Sequester Ribosome
2.Prevent normal AUG from being used on the message

This Slide Property of Jeff Singer

g

 that are used to initiate normal 5ʹ cap-dependent translation (Figure 7–68). I

fact, different IRESs require different subsets of initiation factors. However, all o

IRES
them bypass the need for a 5ʹ cap structure and the translation initiation facto
that recognizes it, eIF4E.

3′

AA
(A) (B)

A
AA
A A AA A AAA 3′
A

AA
AA
eIF4G
• Folds into a distinct structur IRES
poly-A-binding
• Does not need Cap, but does protein 5′ cap
eIF4E eIF4G
need other translation initiation
factor
• Frequently found in Viruses

other translation factors

small and large ribosomal subunits

TRANSLATION INITIATION TRANSLATION INITIATION

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 effect on its degradation (Figure 7–70).

Although poly-A shortening controls the half-life of most
mRNA stability
some mRNAs can be degraded by a specialized mechanism
step altogether. In these cases, specific nucleases
Two mRNA decay mechanisms:
cleave th
Bacteria:
1.Mostly unstable-half lives of several coding sequence 3′ UTR
minute cap
5′ AAAAA~200 3′
2.Exonucleases responsible for thi
3.Allows rapid response to gradual poly-A shortening
environmen
5′ A~25 3′
Eukaryotes decapping followed
by rapid 5′-to-3′ continued 3′-to-5′
1.30 to 600 minute half live
degradation degradation
2.Endonucleases in some case
A~25
3.5’ and 3’ ends 1 2

Can work at the same time as well!

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 mRNA stability
POST-TRANSCRIPTIONAL CONTROLS
The same features are recognized for degradation and translation, thus they
compete:
A Figure 7–70 The
A
A mRNA translatio
A The same two fe
AAAAAAAA 3′ deadenylase
AAAA
AAA molecule—its 5ʹ
AAAAA
eIF4G
tail—are used in
and deadenylatio
poly-A-binding decay (see Figure
protein 5′ cap that shortens the
eIF4E 5ʹ direction assoc
As described in C
6–70), the transla
also associates w
the poly-A tail. (A
et al., Mol. Cell 5
permission from

translation initiation mRNA degradation

This Slide Property of Jeff Singer
 We saw in Chapters 3 and 6 that large aggregates of proteins and nucleic acids

Dual regulation: Aconitase - Fe regulation

that work together are often held in proximity by loose, low-affinity connections
(see Figure 3–36). In this way, they function as “organelles” even though they are
not surrounded by membranes. Many of the events discussed in the previous

IRON STARVATION

cytosolic aconitase cytosolic aconitase

ferritin mRNA transferrin receptor mRNA

5′ AAA 3′ 5′ AAA 3′
translation blocked
Positiv mRNA is stable and translated

– Stabilize mRN (A)

NO FERRITIN MADE TRANSFERRIN RECEPTOR MADE

Negativ
– Repress translation EXCESS IRON
Fe Fe

endonucleolytic
cleavage

ferritin mRNA transferrin receptor mRNA

5′ AAA 3′ 5′ AAA 3′
mRNA translated mRNA degraded

FERRITIN MADE NO TRANSFERRIN RECEPTOR MADE

(B)

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 they differ in the way the short pieces of single-stranded RNA are generated, all

Small non-coding RNAs

three types of short RNAs locate their targets through RNA–RNA base-pairing, and
they generally cause reductions in gene expression.

miRNAs Regulate mRNA Translation and Stability

Another exception to the central dogma:
Over 1000 different microRNAs (miRNAs) are produced from the human genome,
1. Non-coding RNA and that regulates
these appear gene
to regulate at leastexpression
one-third of all human protein-coding genes.
Once made, miRNAs base-pair with specific mRNAs and fine-tune their transla-
2. Pol II transcripts
tion and stability. The miRNA precursors are synthesized by RNA polymerase II
and are capped and polyadenylated. They then undergo a special type of process-
3. RNAs are destroyed
ing, after in large
which complexes
the miRNA called
(typically 23 P-bodies
nucleotides in length) is assembled with
a set of proteins to form an RNA-induced silencing complex or RISC. Once formed,
4. Most regulate the
gene expression via an RNA interference mechanism
RISC seeks out its target mRNAs by searching for complementary nucleotide (RNAi)

interfering RNA target

processing RNA
Figure 7–74 RNA interference in
double-stranded eukaryotes. Single-stranded interfe
RNA Argonaute or Piwi RNAs are generated from double-s
proteins RNA. They locate target RNAs thro
base-pairing and, at this point, seve
are possible, as shown. As describe
the text, there are several types of R
interference; the way the double-str
cleavage of translational repression formation of heterochromatin RNA is produced and processed an
target RNA and eventual destruction on DNA from which target RNA ultimate fate of the target RNA depe
of target RNA is being transcribed the particular system.

This Slide Property of Jeff Singer

 P-bodies
Dcp1a Argonaute Merge

This Slide Property of Jeff Singer

 Small non-coding RNAs
Small Non-coding RNA that regulate gene expression:


Three classes
1. microRNAs (miRNAs )
2. small interfering RNAs (siRNAs )
3. piwi-interacting RNAs (piRNAs )

This Slide Property of Jeff Singer

-

 same mRNA lead to further reductions in its translation. As discussed earlier for
transcription regulators, combinatorial control greatly expands the possibilities

miRNAs available to the cell by linking gene expression to a combination of different reg-
ulators rather than a single regulator. Third, an miRNA occupies relatively little

CLEAVAGE
“CROPPING”

Non-coding RNA that regulates AAAAA

NUCLEUS

gene expression:
 CLEAVAGE

CYTOSOL

“DICING”
Figure 7
ONE STRAND DEGRADED
mechan
miRNA,
Three classes Argonaute and
other proteins 3′ 5′ RISC
one part
forms a
RNA is c
1. microRNAs (miRNAs ) extensive match less extensive match and then
it is furth
2. small interfering RNAs (siRNAs ) mRNA mRNA
to form t
AAAAA AAAAA in conjun
3. piwi-interacting RNAs (piRNAs ) “SLICING”
RISC, in
of the m
discards
guides R
AAAAA
base-pa
ATP extensiv
Argonau
ADP causing
the miRN
RISC released for reuse
extend b
AAAAA “seed” re
miRNA.
leads to
RAPID TRANSLATIONAL REPRESSION destabili
RAPID mRNA DEGRADATION EVENTUAL DEGRADATION OF mRNA P-bodies

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 micro RNAs
Sets of genes that contain inverted repeats, in worms one is called let-7. Conserved across species.

Primary micro RNA

This step involves drosha RNases.

Precursor micro RNA

This step involves several dicer type

RNases, with different substrate
specificity.

The mature micro RNAs can

either block translation or
catalyze mRNA destruction

This Slide Property of Jeff Singer

micro RNA processing

1. Drosha
releases
dsRNA from
micro RNAs
for dicer.

This Slide Property of Jeff Singer

The RISC pathway

2. Dicer releases
short dsRNA
molecules for
RISC

RNA-Induced Silencing Complex, or RISC

This Slide Property of Jeff Singer

 RISC consists of 4 proteins:
AGO2, VIG, FXR and Tudor-SN (TSN) assemble to form RISC.

This Slide Property of Jeff Singer

 Risc protein Functions
1. Ago2-dsRNA helicase

2. VIG (Vasa intronic gene)-an RNA binding protein

3. dFXR or Fmr-Fragile X mental retardation syndrome related protein,

associated with fragile X syndrome. An RNA binding protein.

4. Tudor-SN (TSN)-nuclease, degrades the target mRNA.

This Slide Property of Jeff Singer

 RISC
RISC unwinds dsRNA and uses the ssRNA to direct the complex to
REGULATION OF GENE EXPRESSION BY NONCODING RNAs
the RNA to be modified:
3′ OH (end of RNA)
nucleotides that
search out target RNA

microRNA

5′
phosphate

active site, showing

2 Mg2+ atoms needed for “slicing”
Human Argonaute protein carrying an miRNA.
• The miRNA is held in an extended form that is optimal for forming RNA–RNA
base pairs.
space in the•genome when compared with a protein. Indeed, their smal
The active site of Argonaute is extensively base-paired with the miRNA (red).
one reason that miRNAs were discovered only recently. Although we a
beginning to appreciate the full impact of miRNAs, it is clear that they re
an important part of the cell’s equipment for regulating the expression o
We discuss specific examples MBoC6 This
of miRNAs Slide
that
m7.113/7.77
Property
have of Jeff
key roles Singer
in developm

 many organisms, the RNA interference machinery maintains the heterochroma-

RNA interference can change chromatin structure to cause transcriptional
tin formed around centromeres. Centromeric DNA sequences are transcribed
in both directions, producing complementary RNA transcripts that can base-
pair to form double-stranded RNA. This double-stranded RNA triggers the RNA
repression
double-stranded RNA

This structure recruits Argonaute and

other RISC proteins
siRNAs
Argonaute and
other RITS proteins

chromatin remodeling
factors that can lead to RISC RITS

heterochromatin
formation PATHWAY NOW FOLLOWS ONE OF
THOSE SHOWN IN Figure 7–76

Figure 7–77 RNA inte

by siRNAs. In many o
stranded RNA can trig
RNA polymerase destruction of complem
(left) and transcriptiona
The change in chroma
induced by the bound
HISTONE METHYLATION
DNA METHYLATION induced transcriptiona
RNA-induced transcriptional silencing (RITS) TRANSCRIPTIONAL REPRESSION resembles that in Figur

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siRNA and DNA expressing shRNA can target the degradation of specific
mRNAs in cultured cells

This Slide Property of Jeff Singer

RNAi screens can explore the function of all the genes in cultured cells

This Slide Property of Jeff Singer

RNAi can be used to suppress genes in a tissue specific manner

This Slide Property of Jeff Singer

RNA interference is a defense mechanism

Involved in defense against double stranded RNA such as that is found

in viruses and transposons

This Slide Property of Jeff Singer

 piRNAs
1. A third system of RNA interference relies on piRNAs (piwi-interacting RNAs , named for
Piwi, a class of proteins related to Argonaute).

2.piRNAs are made speci cally in the germ line, where they block the movement of
transposable elements

3.Genes coding for piRNAs consist largely of sequence fragments of transposable elements.
These clusters of fragments are transcribed and broken up into short, single-stranded
piRNAs.

4.Once formed, the piRNAs seek out RNA targets by base-pairing and, much like siRNAs,
transcriptionally silence intact transposon genes and destroy any RNA (including mRNAs)
produced by them.

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fi

Bacteria Use Small Noncoding RNAs to Protect Themselves from Viruses

CRISPR-mediated immunity in bacteria and archaebacteria.
• After infection by a virus (left panel), a small bit of DNA from the viral genome is inserted into the CRISPR locus.
• A small fraction of infected cells must survive the initial viral infection and the surviving cells descendants, transcribe the CRISPR locus and
process the transcript into crrnAs (middle panel).
• Upon reinfection with a virus that the population has already been “vaccinated” against, the incoming viral DNA is destroyed by a
complementary crrnA (right panel).

CRISPR-Clustered regularly interspaced

short palindromic repeats
CAS-CRISPR associated

Using CRISPR as a tool-in upcoming topic

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 example in telomerase, where the RNA molecule holds together and organizes function in the same process. As described
protein components. These RNA-based scaffolds are analogous to protein scaf- in Chapter 6, RNAs can fold into specific

Long Non-coding RNAs

folds we discussed in Chapter 3 (see Figure 3–78) and Chapter 6 (see Figure 6–47).
RNA molecules are well suited to act as scaffolds: small bits of RNA sequence,
often those portions that form stem-loop structures, can serve as binding sites
three-dimensional structures that are often
recognized by proteins. (B) In addition to
serving as scaffolds, lncRNAs can, through
formation of complementary base pairs,
for proteins, and these can be strung together with random sequences of RNA localize proteins to specific sequences on
Roles of long noncoding RNA (lncRNA)
in between. This property may be one reason that lncRNAs show relatively little RNA or DNA molecules. (C) In some cases,
(A) lncRNAs can serve as scaffolds, bringing together proteins that function in the same process lncRNAs act only in cis, for example,
primary-sequence conservation across species.
(B) In addition to serving as scaffolds, lncRNAs can, through formation of complementary base pairs, localize when the
proteins to RNA
speciis cheld in place by
sequences onRNA
RNA or DNA
The second key feature of lncRNAs is their ability to serve as guide sequences, polymerase (top). Other lncRNAs, however,
molecules.
binding to specific RNA or DNA target molecules through base-pairing. By doing diffuse from their sites of synthesis and
(C) In some cases, lncRNAs
so, they bringactproteins
only in cis,
thatforare
example,
boundwhen the RNA
to them intoisclose
held in place by RNA
proximity withpolymerase
the DNA (top). Other act
therefore lncRNAs,
in trans.however, diffuse from their
sites of synthesis and therefore act in trans.

Telomerase
controls transcription of ACTS IN CIS
genes on same chromosome

Xist
IncRNA
(A) IncRNA
chromosome A
RNA polymerase
controls transcription of
genes on other chromosomes,

ACTS IN
TRANS
IncRNA

RNA DNA chromosome A chromosome B

(B) (C)

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fi
 Post-transcriptional regulation
• Transcriptional Attenuation – Leaky scanning/uOR
and Riboswitche – IRE

• Alternative splicin – mRNA stabilit

• mRNA cleavag • Small non-coding RNA

• RNA editin • Long non-coding RNAs
• Nuclear expor
• mRNA localizatio
• Translational contro
– eIF-
This Slide Property of Jeff Singer
S

 Next:

Topic 28: Protein Methods

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