Regulating genes by

RNA
Eukaryotic RNA regulatory mechanisms
 Learning Objectives
• Understand how exons and introns are marked in
the nucleus
• Learn the concept of alternative splicing and the
contribution it makes to genome complexity
• Understand the importance of RNA regulatory
regions in mRNA stability and translation
• Learn how RNA interference protects the cell
from viruses and transposable elements
• Learn how RNAi can be exploited in gene
knockdown
• Understand how RNA technology can be used
for gene therapy
Lewin. Essential Genes (2006)
 SR proteins recruit spliceosome
components to the 5’ and 3’ splice
sites

Molecular Biology of the Gene

 Alternative splicing
• Many primary transcripts can be spliced in
alternative ways to generate different
mRNAs and thus different protein products
(or isoforms)
• 40% of Drosophila genes and as many as
75% of human genes undergo alternative
splicing
• In some cases, hundreds (e.g the human Slo
gene) or even thousands (for the Drosophila
Dscam gene) of potential alternatives can
be generated.
The Drosophila Dscam gene
RNA regulatory mechanisms
• In addition to regulation by splicing, RNA expression
can be regulated at the level of stability and translation
• E.g. regulation of iron levels in the cell
• Iron is delivered to the cell by the cell surface
transferrin receptor
• Inside the cell, toxic iron is rendered safe by the
binding protein ferritin
• Both the transferrin receptor and ferritin mRNAs are
subject to regulation by Iron response elements
• Transferrin receptor mRNA is degraded in the
presence of iron
• Ferritin mRNA cannot be translated in the absence of
iron
• The IRE binding protein is aconitase, the TCA
enzyme that requires iron as a cofactor for activity
Regulatory RNA in eukaryotes
• Recent estimates based on transcriptomic and
bioinformatic analysis suggest that there may be
thousands of non-coding RNAs in the human genome
• More than 75% of the genome is transcribed
• some of these are well known,
• e.g. tRNAs, rRNAs, snRNAs, snoRNAs
• In addition, eukaryotic genomes code for many short (~22
base) RNA molecules called microRNAs (miRNAs).
• Involved in RNA interference (RNAi) – A process by
which short (21 to 23) nucleotide antisense RNAs,
derived from longer double-stranded RNAs, can modulate
expression of mRNA by translation inhibition or
degradation.
 RNA Interference
• miRNA – Genome derived RNA sequences transcribed in the nucleus and
processed from longer precursors.
• piRNAs regulate gene expression in germ cells and act to silence
transposable elements.
• siRNAs are derived from dsRNA in the cytoplasm and are complementary
to viruses and transposable elements.
• Generated from longer precursors by:
• Drosha – A nuclear endonuclease that processes double-stranded
primary RNAs into short, ~70 base pair precursors for Dicer processing.
• Dicer – An endonuclease that processes double-stranded precursor RNA
to 21 to 23 nucleotide RNAi molecules.
• RNA complexed by RISC – RNA-induced silencing complex, a
ribonucleoprotein particle composed of a short, single-stranded RNA
and a nuclease that cleaves mRNAs complementary to the siRNA or
miRNA.
• It receives RNA from Dicer and delivers it to the mRNA.
How does RNAi work?
• MicroRNAs regulate gene expression by
base pairing with complementary sequences
in target mRNAs.
• RNA interference triggers degradation or
translation inhibition of mRNAs
complementary to miRNA or siRNA.
• It can also lead to mRNA activation.
• Viral genomes can fight back with their
own miRNAs
 Gene knockdown
• Experimentally exploit the RNAi pathway
to specifically target mRNAs
• A convenient and quicker way to inhibit
gene activity than using mutants or knock
outs
• Cells can be transfected with siRNA made
in vitro
• Transgenic animals or transfected cells can
be made to express dsRNA or shRNA from
transgenes, that will be processed by Dicer
from transcribed precursors
Lisa J Scherer & John J Rossi
Nature Biotechnology 21, 1457
- 1465 (2003)
 RNA therapy?
• Can the RNAi pathway be exploited to
manipulate disease gene expression?
• Principle problem = delivery
• 2 solutions
• Viral delivery using shRNA constructs
• Chemical delivery using reagents that will
• neutralize the -ve charge of the RNA
• allow interaction with and passage
through the plasma membrane
 The Bottom Line

• RNA based regulatory mechanisms are an

important component of gene regulation
• RNAi gene knockdown is cheaper and more
convenient than gene knockout