Interference RNA Pathways, RNAi
A set of related processes that shut down gene
expression by one of three different mechanisms:
Translational repression, mRNA degradation, and
heterochromatin formation.
All are mediated by small RNA molecules that use
their sequence to target particular mRNAs and
genes and which are generated by a common
enzymatic pathway.
RNA Interference was first discovered in worms and
plants, but forms of it are now known to exist in all
metazoans and many unicellular eukaryotes.
Heterochronic mutations in the worm C. elegans alter the timing of
developmental transitions in the larvae. The Lin-4 gene represses
expression of the Lin-14 protein in the early larva. The let-7 gene
represses Lin-41 later in development.
Work by Victor Ambros and Gary Ruvkin
Banerjee D, Slack F. Bioessays. 2002;24(2):119-29.
Instead of encoding proteins, the lin-4
and let-7 genes produced very small 21
nt. RNAs derived from larger stem-loop
structures.
Banerjee D, Slack F. Bioessays. 2002;24(2):119-29.
The short Lin-4 RNA can base pair to the 3 UTR of its target Lin-14,
thereby repressing its translation. Let-7 similarly binds and represses
its target genes.
Banerjee D, Slack F. Bioessays. 2002;24(2):119-29.
Different forms of gene silencing in plants and worms
turned out to use similar enzymatic machinery.
RNA interference
in worms causes
degradation of target
mRNA
Cosuppression
in plants causes
gene silencing
through heterochromatin
formation
Target gene
Clone inverted repeat
of target gene
Expressed as
dsRNA
hairpin
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The generation of Lin-4 and Let-7 microRNAs requires the same
gene products as RNA Interference and cosuppression. All of
these processes are mediated by very short (21-26 nt) RNAs
processed out of a longer double stranded RNA precursor.
Chromatin Silencing has
been primarily studied in
plants and some fungi
and is less understood
mechanistically.
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3
We will discuss the
mRNA degradation and
translational repression
mechanisms.
Chromatin
Silencing
mRNA
Degradation
By siRNAs
Translational
Repression
By miRNAs
Long double stranded RNA is cleaved by the enzyme Dicer into short
duplexes of 21 nt RNAs with overhanging 3 ends.
One strand of this
duplex is transferred
into the RNA Induced
Silencing Complex or
RISC.
These RNAs are
called short
interfering RNAs, or
siRNA.
If they are perfectly
complementary to a
target mRNA, a
subunit of RISC
called an Argonaute
protein will cleave the
target mRNA.
Endonucleolytic cleavage of an mRNA will lead to its rapid destruction
by both 5-3 and 3-5 exonucleases.
The RNA degradation arm of the RNAi pathway serves to degrade
certain viral RNAs and to limit expression from transposable elements.
This process is now a key experimental technique for knocking out
gene function and is the basis for numerous experimental therapeutics.
In mammalian cells, long dsRNA induces the interferon response.
Interfering RNAs are delivered into cells simply as short dsRNA that
can be directly loaded into RISC, or can be expressed from a gene on
a plasmid that will produce a short hairpin to be processed by Dicer.
siRNA
Transfection
into cells
PTB siRNA:
N2A Cells
-
+
PTB
shRNA
mm
I
GAPDH
MicroRNAs such as Lin-4 and Let-7 are expressed from stem loop
precursors that are processed by Dicer. They are not completely
complementary to their target mRNAs and induce translational repression
but not usually direct mRNA cleavage.
Lin-4 was the first example of a microRNA (miRNA). MiRNAs bind to
multiple sites in a target mRNA repress to its translation. Unlike
siRNAs, miRNAs do not form perfect duplexes with their target and do
not generally induce cleavage.
He L, Hannon GJ.
Nat Rev Genet. 2004;5(7):522-31.
If the short RNA loaded into Argonaut is perfectly complementary to
its target, it will induce RNA cleavage. If the small RNA forms
internal bulge loops with the mRNA, as seen with the Lin4 and Lin14
miRNAs, translation is usually repressed without mRNA cleavage.
In this case it is important that the first 8 nucleotides of the miRNA
(the seed region) be almost entirely paired.
The primary transcripts for
microRNAs (pri-miRNAs) are
Pol II transcripts. miRNAs
may be encoded in the
introns or UTRs of protein
encoding transcripts, or can
have a transcript of their
own. Different microRNAs
can be clustered on the
same transcript. These are
often involved in a common
pathway of regulation.
Du T, Zamore PD. Development. 2005;132(21):4645-52.
Vertebrates express hundreds of microRNAs (miRNA) similar to
Lin-4 and Let-7. They can show highly tissue specific expression
and control particular genetic pathways.
MicroRNAs showing region specific expression
in the Zebrafish brain.
Wienholds, E., and R.H.A. Plasterk; FEBS Letters, 2005; 579:5911-5922
Individual miRNAs are involved in many different cellular processes,
including growth control, cell death, and cancer. Many are needed for
controlling specific developmental pathways and the differentiation of
specific cell types.
Mouse Forelimb Development
Dicer Mutant
Wildtype
Harfe BD, et al Proc Natl Acad Sci U S A. 2005;102(31):10898-903.
Mice lacking miR-1.2 exhibit ventricular septal defects and hyperplasia
of cardiomyocytes. Products from several miR-1.2 target mRNAs are
upregulated, including the heart specific transcription factor Hand2.
Zhao et al. Cell. 2007;129(2):303-17.
The primary miRNA
transcript is cropped in
the nucleus by the
enzyme Drosha, and
exported by Exportin 5.
In the cytoplasm, the
pre-miRNA hairpin is
processed to a 21nt
duplex by Dicer (the
same enzyme that
processes siRNAs). The
short duplexes are
unwound and one strand
is loaded into RISC,
which contains an
Argonaute protein.
These RISC complexes
are targeted to
complementary mRNAs
by the loaded miRNA.
He L, Hannon GJ.
Nat Rev Genet. 2004;5(7):522-31.
Both Drosha and Dicer contain double-stranded RNA binding
domains (dsRBD), and RNase III domains, which can cleave both
strands of an RNA duplex. Dicer also contains a PAZ domain, which
will bind to RNA ends. The dsRBD may act as a ruler to determine
the 21-22nt length of the product.
Drosha
Dicer
Hammond SM. FEBS Lett. 2005;579(26):5822-9.
Argonaute Proteins have two domains, PAZ and PIWI. The PAZ domain
will bind to the end of the miRNA. The PIWI domain can have nuclease
activity, depending on the Argonaute.
Hammond SM. FEBS Lett. 2005;579(26):5822-9.
Drosha and Dicer each have
cofactors that are required for
their activity. Drosha forms a
complex with a nuclear protein
called DGCR8 or Pasha. Dicer
forms a complex with TRBP.
Wienholds, E., and R.H.A. Plasterk;
FEBS Letters, 2005; 579:5911-5922
The mechanism of translational repression by RISC
is still unclear. The binding of the RISC seems to
direct the mRNA into P-bodies.
Yet another branch of the RNAi system are the piRNAs.
- 29 nt RNAs bound by Piwi protein.
- Germline specific expression
- Suppress transposon expression and transposition
in germline.
-Generated at specific loci from sequences related to
transposons in Drosophila flamenco
- other loci in mammals