Lecture 20

Eukaryotic gene regulation:

Control at the level of RNA

Epigenetics
changes in patterns of gene expression that occur
without changes in the DNA sequence
Lectures 18 and 19: modifications of histones,
modifications of DNA passed on through mitosis and
meiosis
This lecture: the role of RNA in gene expression
Relates to lecture 10: How researchers use activities
of RNA molecules to alter gene function (RNAi)
RNA may mediate many epigenetic effects
(e.g., Xist)

Centraldogma:
DNARNAproteinphenotype

Six steps at which eukaryotic gene expression can be controlled.

(Alberts, Fig. 9.2)

General mechanism of ribonucleases and

small self-cleaving ribozymes.
The 2'-hydroxyl adjacent to the scissile phosphate is activated for
nucleophilic attack by abstraction of its proton. Concurrently, a
proton is donated to stabilize the developing negative charge on the
leaving group oxygen.
-- Doudna and Cech. 2002. Nature 418: 222-227.

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

Alternative RNA splicing:

many proteins from a single mRNA
Increases number and diversity of
eukaryotic proteins
Involved in cell type specificity of gene
expression

What does RNA splicing do?

Alternate splicing allows one mRNA to encode

(multiple) different polypeptides with different activities

http://www.nature.com/horizon/rna/

Alternative splicing of the Drosophila Dscam gene can generate

up to 38,016 different isoforms from this one gene.

A more typical
eukaryotic gene has 2
introns and 3 exons
(avg for human genes
9 exons of total length
3.4 kb, the median
numbers are 7 introns
of length 1.0 kb)
An estimated 75% of
human genes show
some experimental
evidence of alternative
splicing

The major signals for splice site recognition

are near the ends of each intron.

Unexpected steps in splicing reaction: lariat formation

as splicing intermediate

Spliceosome holds components together

during mRNA splicing reaction
Spliceosome contains crucial RNAs:
snRNAs (~ 5 RNAs), 100-300 nt long
Some base pair with
splice donor and acceptor
~50 proteins involved
Because no net energy is required,
no protein enzymatic activity is involved
in mRNA splicing
However, proteins are involved likely
for complex assembly & rearrangement, and
RNA helicases - ATP is utilized

Exon recognition.
- 5' (GU) and 3' (AG) splice sites near exons are recognized by the splicing machinery
- Exons contain exonic splicing enhancers (ESEs)
- ESEs are bound by SR proteins.
- SR proteins recruit U1 snRNP to the downstream 5' splice site
U2AF (65 and 35 kDa subunits) to the pyrimidine tract (YYYY) &
the AG at the upstream 3' splice site
- U2AF recruits U2 snRNP to the branchpoint sequence (A)
- Bound SR proteins recruit splicing factors to form a 'cross-exon' recognition complex.
- SR proteins also function in 'cross-intron' recognition by facilitating the interactions
between U1 snRNP bound to the upstream 5' splice site and U2 snRNP bound to the
branchpoint sequence.
-- Maniatis and Tasic. 2002. Nature 418: 236-243.

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

Sex determination in Drosophila controlled by differential

transcription initiation and differential splicing

Sxl transcription in females

from
early promoter (Pe)
in early embryos
generates Sxl protein

Sxl alternative splicing results in protein only

in females
IN FEMALES

Low level of Sxl protein binds

exon 3 on the RNA
Sxl is a sequence-specific
RNA-binding protein
Sxl binding blocks splicing
Exon 3 is skipped, producing
mRNA that encodes functional
protein
Sxl promotes female
development by regulating
expression of female-specific
genes
Regulation at RNA level

Sxl alternative splicing results in protein only

in females
IN MALES

No initial Sxl
Splicing includes exon 3
Stop codon truncates
protein
No functional Sxl
Female traits not
activated

Sxl regulation

Initial differences set up by differential transcription of

an RNA binding protein (Sxl) in females
Sxl then regulates alternative splicing of Sxl in females
Unregulated splicing produces an RNA with a stop
codon yielding a truncated Sxl in males
Sxl sequence-specific binding to RNA results in
alternative splicing and production of active Sxl
Sxl regulates females differentiation via a cascade of
downstream differential splicing events
More details in Chapter 16.5

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

Catalytic properties of RNA the RNA

world

Self-splicing is mediated by RNA alone

Cechs lab studying very basic biology in Tetrahymena

thermophila
Observed that pre-rRNA includes intron
Totally unexpectedly from control experiment it was
found that in vitro, the pre-rRNA spliced correctly without
any protein added to the reaction
First example that RNA can function as a catalyst, like
enzymes, without protein = ribozyme
aka catalytic RNA

Tetrahymena self splicing mechanism

Group II: (mt and cp RNAs)
A in intron attacks 5 splice
site to form lariat

Reaction proceeded without protein!

Lodish, Mol Cell Biol

Tetrahymena splicing mechanism same as

pre-mRNA splicing

Lodish, Mol Cell Biol

Model: mRNA splicing mechanism may have

evolved from Group II introns

5 snRNAs involved in
pre-mRNA splicing form
secondary structure
similar to group II introns
snRNAs that mediate premRNA splicing may be
related to Group II introns
(now separated and
acting in trans)
Proposal: mRNA splicing
arose from ancestral selfsplicing

Black arrow = 1st reaction

Blue arrows = 2nd reaction
Alberts, Mol Biol Cell

Ribozymes designed to cleave RNA of

interest

Catalytic core

Biochemical Journal (2004) 379, 823-832 www.biochemj.org

Ribozymes as tools to knock down

gene expression

Ribozymes base pair with target RNA

Separate catalytic core mediates cleavage
Potential to cleave specific RNAs highly
specific!
Used for basic and clinical research

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

RNA editing discovered in protozoa

mt DNA

Trypanosoma, Leishmania and other parasitic

protozoa
Parasites, disease causing
Single cell eukaryotes

Trypanosome mt DNA

Single mitochondrion = kinetoplast

kDNA = network of minicircles and maxi circles
Minicircles: 10-25,000 each 0.5-2.5 kb
Maxicircles: 50-100, 21-31 kb
Minicircles
Maxicircles

Kinetoplast

RNA editing discovered in

trypanosomes

kDNA encodes mRNAs that are processed

Maxicircles contain protein coding genes
Minicircles: no protein coding genes
Yet.Initial sequencing revealed paradox:
Transcripts were found from maxicircles
DNA sequence = only short gene fragments
RNA sequence included both fragments
and protein-coding RNAs

RNA editing discovered in

trypanosomes

RNA editing: alters maxicircle RNAs to

generate full-length coding regions
Unedited, RNAs would not encode proteins
(e.g., lack start or stop codon or contain
incorrect codons)
Editing requires RNA made from minicircles

What is RNA editing?

Insertion and deletion of U and C residues

Base modification by deamination (C to U, A to I)
Proposed to represent primordial mechanisms in
RNA world

RNA editing: editosome includes

proteins and guide RNA

Proteins/enzymes to catalyze
removal or addition of Us,
RNA ligation

1000s of small gRNAs

encoded by minicircles
gRNAs include:
anchor that base pairs with
pre-mRNA
Information that will be
incorporated into the premRNA

Adapted from B. Bass, 1993, in R. Gesteland and J. Atkins, eds., The RNA World

RNA editing found in humans!

ApoB (C to U change)
Cell-type specific expression of
B48 vs. B100
coronary heart disease

GluR2 (A to I; Q to R)
GluR2(Q) Ca2+ permeable
GluR2(R) Ca2+ impermeable
sporadic ALS w/ defect in
editing in motor neurons

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

DNA --> RNA --> protein --> phenotype

translation control

Kozak sequence: (gcc)gccRccAUGG

R: purine A or G

The elongation phase of translation

This three-step cycle is
repeated over and over during
synthesis of a protein chain.
1. An aminoacyl-tRNA binds
to the A-site on the
ribosome
2. A new peptide bond is
formed and
3. The ribosome moves a
distance of three
nucleotides along the
mRNA chain, ejects an old
tRNA molecule and
"resets" the ribosome so
the next aminoacyl-tRNA
molecule can bind.
The P-site is shown on the left
side of the ribosome, with the
A-site on the right.
(Alberts, Fig. 6,.22)

The binding of release

factor to a stop codon
terminates translation.
The completed
polypeptide is
released, and the
ribosome dissociates
into its two separate
subunits.

Termination of translation.

(Alberts, Fig. 6.24)

Regulation of Translation
Global: Regulation of translation proteins: phosphorylation of eIF2
(eukaryotic initiation factor 2) responds to adverse cellular states to inhibit
translation initiation (some viruses take advantage of this)
Specific: Secondary structure of mRNA can influence the efficiency
of translation of specific transcripts (e.g., in UTRs)
Initiation:
Ribosome binding sites (RBS)- start codon context
#s of ribosomes recruited (monosome vs. polysomes, ribosome profiling)
Upstream open reading frames (false starts)
Elongation:
Codon optimality affects speed of translation
Ribosomal frameshifting- pseudoknots -1, slippery sites (UUUAAAC) +1
Termination:
Translational read through
- Nonsense suppressor tRNAs mutation in anticodon (Ec, Sc, viruses)
- Programmed translational readthrough (VEGF-Ax, Hs colon cancer)

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- Ribozymes
- RNA editing
- Translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

DNA --> RNA --> protein --> phenotype

RNA stability

Mechanisms of RNA
Degradation
From most common to
least:
loss of the polyA tail
decapping
exonuclease degradation

http://kiledjianlab.cbn.rutgers.edu/

AU-rich element (ARE) mediated decay

AU-rich sequences
(clusters of AUUUA motifs)
in 3UTR of short-lived
mRNAs of cytokines, TFs,
proto-oncogenes
Most common determinant
of instability for genes in
mammalian cells.
After van Hoof and Parker. 2002.
Current Biology 12: R285-R287

ARE-binding protein (purple oval) bound to its 3' UTR.

Recruits Poly(A) nuclease (green pacman). The exosome (blue ovals).
Decapping activity (orange pacman) degrades the 7mGpppN cap structure
Independent of siRNA/miRNA degradation pathways

Nonsensemediated decay
(NMD) is the
degradation of
mRNAs carrying
premature stop
codons.
Important for
turnover of nonfunctional products
of regulated
splicing (e.g. Sex
lethal in males).

Alternative splicing by spliceosomes requires

specific sequences only in the ___
A.
B.
C.
D.

exons
Introns
exons and introns
3 UTRs

Cis-acting sequences are not important

for the regulation of translation.
A.
B.

True
False

Outline:
- Splicing
- Sex determination in Drosophila (an example)
- RNA editing
- Export from the nucleus
- Translation and regulation of translation
- RNA stability
- small RNAs: siRNAs and miRNAs
- RNA localization
- protein degradation

Antisense RNA as tool to

knockdown gene expression
Tested for many
applications
Basic research
Clinical

RNAi knockdown of GFP reporter

Cells respond to virus attack by

destroying viral RNA

ds RNA in cell
activates:
Anti-viral response
Response to
transposons
Enzymes chop up ds
RNA and then chop
up matching viral
mRNA

RNAi destroys mRNA

Dicer and RISC (RNA-induced silencing
complex).
-RNAi is initiated by the Dicer enzyme,
which processes double-stranded RNA into
22-nucleotide small interfering RNAs.
-siRNAs are incorporated into a
multicomponent nuclease, RISC (green).
-RISC is believed to be latent when
containing a ds siRNA and unwinding of
siRNAs, activates RISC*.
-RISC* then uses the unwound siRNA as a
guide to substrate selection.

RNAi
Systemic effects: Feeding dsRNA to C. elegans results in
RNAi in other tissues, same in plants.
Systemic effects require spreading of the dsRNA via an RNAtransporter
Transporters SID-1 vs. SID-2 allow dsRNA to enter new cells
(C.elegans)
RNA-dependent RNA polymerase amplifies signal (worms
and plants)
Transgenerational inheritance of RNAi: Injecting adult
females with dsRNA produced defects in their offspring (in
beetles); (worms and plants)
heritable epigenetic effects

Other classes of small RNAs (20-30 nt)

with distinct activities
siRNA- small interfering RNAs (generally
from exogenous RNA like dsRNA
introduced by RNAi or from viruses)
piRNA PIWI interacting RNAs
(generated from genomic regions with
repetitive sequences), repress
expression of transposable elements
miRNA- micro RNAs (transcribed from
endogenous genes)

Micro-RNAs
New class of regulatory RNA
Small RNAs that do NOT encode
proteins (ncRNAs)
~ 25% generated from introns
Most transcribed by RNA pol I
Natural RISC complex RNAs
~5600 miRNAs in the human genome,
56.7% specific to humans (Londin et al., 2015)
Many show tissue-specificity

How are miRNAs generated?

60-120 base stem loops trigger processing

Drosha RNase binds stem loop, cleaving at base
Pre-miRNA transported to cytoplasm
Dicer RNase trims ends to produce 12-24 nt miRNA duplex
Duplex miRNA incorporated into RISC (RNA-induced Silencing Complex)
One strand degraded to yield single strand RNA loaded into RISC
miRNA now active and ready to degrade other cellular RNAs

miRNA mediated RNA interference

If miRNA finds perfect

match in cellular mRNA

miRISC binds mRNA

Cleaves bound mRNA
mRNA halves no longer
protected from cellular
RNases
mRNA degraded
miRISC is recycled to
target more mRNA

miRNA mediated RNA Interference

If miRNA finds
imperfect match in
cellular mRNA

miRISC binds mRNA

Cannot cleave
Inhibits progression of
ribosomes
Translation of mRNA
decreased

RNA-directed DNA methylation (RdDM).

In A. thaliana, dsRNA can be produced by RNADEPENDENT RNA POLYMERASE 2 (RDRP2) activity on
ssRNA templates or by transcription of inverted DNA repeats
by a DNA-dependent RNA polymerase such as RNA
polymerase II.
dsRNA are processed into RNA signals by DICER-LIKE
enzymes. The RNA signals (wavy red lines) target sitespecific methyltransferases to catalyse de novo methylation
of DNA.

Figures from Matzke

& Birchler, Nature
reviews

Methylation of CGs and CNGs can be maintained to some

extent without the RNA trigger
Long-term maintenance of CG methylation requires the
activity of the histone deacetylase HDA6 and the SNF2-like
protein DECREASE IN DNA METHYLATION 1 (DDM1). CNG
methylation is accompanied by H3K9 methylation

RNA interference-mediated
heterochromatin assembly.
RNA polymerase IV (found in plants)
transcribes small RNAs from tandem
repeats or multiple copies of transposons
in heterochromatin.
The dsRNA is cleaved by Dicer to
produce rasiRNAs (repeat associated
small interfering RNAs).
The siRNAs (in a RISC type complex)
guide histone methyltransferases (HMTs)
to the chromatin to modify histone H3 on
lysine 9 (H3K9).
The methylated form of H3 is bound by
Swi6 or HP1, which also associates with
the methyltransferases, to maintain the
silenced state.

DNA --> RNA --> protein --> phenotype

mRNA localization

The importance of the 3' UTR in subcellular localilzation.

Alberts 9-82

Early embryogenesis depends on RNA localization

bcd protein

(Gilbert Developmental Biology, et al. Fig 9-21 and Harwell

21.18).

RNA localization is surprisingly common

DNA --> RNA --> protein --> phenotype

protein stability

Ubiquitin is added to proteins

targeted for degradation.
These are then brought to the
proteasome for degradation.
Ubiquitin is recycled.

From "Biochemistry and Molecular

Biology of Plants" by Buchanan,
Gruissem and Jones. 2000. ASPB.