Biochemistry of RNA interference Numerous studies have investigated the biochemical mechanisms that underpin RNAi induced gene silencing (Tabara et al., 1999; Mourrain et al., 2000; Sijen et al., 2001). These studies have revealed that RNAi suppresses gene function by promoting degradation of specific mRNA involving highly specific and complex protein–protein interactions that occur in the RNA-induced silencing complex (RISC). Depending on the thermodynamic stability of the 5′-end, both the sense and antisense regions of a given siRNA can enter the RISC complex. However, the antisense strand of the siRNA, which is complementary to the target mRNA, serves to accurately identify the target mRNA and induces sequence-specific degradation in association with other components of RISC at the relatively thermodynamically unstable 5′-end. A key component of RISC is the protein argonaute-2 that binds to a single strand of siRNA. Argonaute-2 and the 5′ strand of the siRNA mediate the recognition of the target mRNA and, with other components of RISC, induce mRNA cleavage with consecutive suppression of protein translation
http://www.ambion.com/techlib/misc/siRNA_finder.html Target prediction The secondary structure of mRNA not only influences the maturation of pre-mRNA and the translation into protein (de Smit&van Duin, 1990; Balvay et al., 1993), it also determines the efficacy of a complimentary siRNA to access its mRNA target (Holen et al., 2002; Kretschmer-Kazemi Far & Sczakiel, 2003). Notably Heale and collegues have developed a secondary structure prediction model to identify nonaccessible mRNA sites for RNAi (Heale et al., 2005). For effective gene silencing engineering of 21-nt doublestranded siRNA with a 2-nt deoxythymideine (Ts) overhang at the 3′-end has been recommended by several groups (Chiu & Rana, 2002; Elbashir et al., 2002; Paddison et al., 2002; Khvorova et al., 2003; Reynolds et al., 2004; Ui-Tei et al., 2004), because a 3′-end overhang is more efficient in guiding dsRNA to unwind. Generally synthesised siRNA should not target introns, the 5′- and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). Furthermore, the guanine (G)–cytosine (C) content of the designed siRNA should be between 30% and 50% and the 5′-ends of antisense and sense strand should have high and low thermodynamic stability, respectively. Investigators should avoid internal repeats and palindromes of siRNA. At certain positions in the sense strand of the 21-nt siRNA, base preferences may be considered: an adenosine (A) at positions 3 and 19; absence of G or C at position 19; and a uracil (U) at position 10; and absence of G at position 13. Indeed, thermodynamic properties of siRNA are critical in determining its stability and gene silencing efficacy (Khvorova et al., 2003). Finally, a BLASTsearch of the appropriate genome database should be performed and low-stringency sequences should be avoided to ensure that no other unrelated genes are targeted to minimize off-target effects. Many effective and specific siRNA have been published already and can be found in the public domain.
Generation of siRNA for silencing of gene expression. (A) From top to below, chemically synthesized siRNA, long dsRNA that can be cleaved by Dicer to form siRNA, and shRNA that can be cleaved by Dicer to form siRNA. (B) From top to below, sense and antisense strands are expressed by RNA polymerase III promoter (e.g., U6 promoter) separately and form a double-stranded siRNA molecule, shRNA are expressed by RNA polymerase III promoter (e.g., U6 promoter) first and then cleaved by Dicer to form mature siRNA. Chemically synthesized siRNA, shRNA, and long dsRNA have been used to generate siRNA by introducing these molecules into cells. After entry into the cytoplasm, shRNA and long dsRNA are cleaved into 21-nt long mature siRNA by a RNase III (Dicer), which is an end-recognition endonuclease). These methods generally result in temporary silencing effects. However, long dsRNA can also elicit responses of the innate immune system such as interferon (IFN) release. To obtain stable and inducible RNAi, researchers have recently developed shRNA structures driven by U6 or H1 promoters (RNase III promoters), wherein the shRNA has 2 short duplex stems: one stem connected to a loop sequence, and the other ending with 6 or more thymidines (T) as the termination signal.
miRNA (micro-RNA)http://en.wikipedia.org/wiki/MiRNA A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long. It is thought to regulate the expression of other genes. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein.The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA). In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
Double-stranded RNA triggers processed into siRNAs by enzyme RNAseIII family, specifically the Dicer family Processive enzyme - no larger intermediates. Dicer family proteins are ATP-dependent nucleases. These proteins contain an amino-terminal helicase domain, dual RNAseIII domains in the carboxy- terminal segment, and dsRNA-binding motifs They can also contain a PAZ domain, which is thought to be important for protein-protein interaction. Dicer homologs exist in many organisms including C. elegans, Drosphila, yeast and humans Loss of dicer: loss of silencing, processing in vitro Developmental consequence in Drosophila and C. elegans Dicer is a conserved protein
The initiation pathway may be amplified by the cell through the synthesis of a population of secondary siRNAs using the dicer-produced initiating or primary siRNAs as templates. These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).
miRNA & siRNA
Origins and Mechanisms
of miRNAs and siRNAs
Present by: Mozhdeh Mirahadi
During RNAi Double-stranded RNAs cut into short double-stranded
RNAs, s(small) i(interfering) RNA's, by an enzyme
called Dicer. These then base pair to an mRNA through a
dsRNA-enzyme complex. This will either lead to
degradation of the mRNA strand
Highly specific process
Very potent activity
So far only been seen in eukaryotes
Evidence 30% of genome is regulated by RNAi
RNA interference (RNAi) is a mechanism that inhibits gene
expression at the stage of translation or by hindering the
transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
Need for interference
Defense against Infection by viruses, etc
As a defense mechanism to protect against transposons and other
Genome Wide Regulation
RNAi plays a role in regulating development and genome
30% of human genome regulated
Silencing in Cenorhabditis elegans
dsRNA administrated to worms
can permeate and affect the entire
body causing a systemic RNA-interference
RNAi studies represents a means
of identifying partial or complete
possibly leading to the
identification of gene function.
RNAi can be induced in C. elegans in three simple ways:
Injection of dsRNA into the worm gonads
Soaking the worms in dsRNA solution
Feeding the worms engineered bacteria producing dsRNA
The Players In Interference
siRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding genome
RNA induced Silencing Complex, that cleaves mRNA
Dicer : produces 20-21 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin in to form pre miRNA; which is later
processed by Dicer
• Small interfering RNAs that have an integral role in the phenomenon
of RNA interference (RNAi), a form of post-transcriptional gene
• RNAi: 21-25 nt fragments, which bind to the complementary portion
of the target mRNA and tag it for degradation
• A single base pair difference between the siRNA template and
the target mRNA is enough to block the process.
• Each strand of siRNA has:
• a. 5’-phosphate termini
• b. 3’-hydroxyl termini
• c. 2/3-nucleotide 3’ overhangs
2-nt 3' overhangs ( UU overhangs )
G/C content: 30-50%.
No basepair mismatch
Synthesised siRNA should not target introns, the 5′and 3′-end
untranslated regions (UTR), and sequences within 75 bases of the
start codon (ATG).
BLAST : eliminate any target sequences with significant homology
to other coding sequences.
Originate from capped & polyadenylated full length precursors (pri-miRNA)
Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA)
Difference between miRNA and siRNA
Function of both species is regulation of gene expression.
Difference is in where they originate.
siRNA originates with dsRNA.
siRNA is most commonly a response to foreign RNA (usually viral) and is
often 100% complementary to the target.
miRNA originates with ssRNA that forms a hairpin secondary structure.
miRNA regulates post-transcriptional gene expression and is often not
100% complementary to the target.
And also miRNA help to regulate gene expression, particularly during
induction of heterochromatin formation serves to downregulate genes pre-transcriptionally
(RNA induced transcriptional silencing or RRIITTSS)
RNase III-like dsRNA-specific ribonuclease
Enzyme involved in the initiation of RNAi.
It is able to digest dsRNA into uniformly sized
small RNAs (siRNA)
Dicer family proteins are ATP-dependent
Rnase III enzyme acts as a dimer
Loss of dicer→loss of silencing processing in
Dicer homologs exist in many organisms
including C.elegans, Drosphila, yeast and
humans (Dicer is a conserved protein)
RISC is a large (~500-kDa) RNA-multiprotein
triggers mRNA degradation in
response to siRNA
Unwinding of double-stranded
siRNA by ATP independent
The active components of an RISC
are endonucleases called argonaute
proteins which cleave the target
Summary of Players
Drosha and Pasha are part of the “Microprocessor” protein
Drosha and Dicer are RNase III enzymes
Pasha is a dsRNA binding protein
Exportin 5 is a member of the karyopherin
nucleocytoplasmic transport factors that requires Ran and
Argonautes are RNase H enzymes
Figure 1. Core Features ooff mmiiRRNNAA aanndd ssiiRRNNAA SSiilleenncciinngg 22
Argonaute: At the Core of RNA Silencing
The Argonaute superfamily can be divided into
three separate subgroups:
the Piwi clade that binds piRNAs,
the Ago clade that associates with miRNAs and
third clade that has only been described thus far in
Figure 2. A Diversity ooff ssiiRRNNAA SSoouurrcceess 24
RISC Assembly and siRNA Strand Selection
Although single-stranded siRNAs can load directly into
purified Argonaute proteins, the double-stranded siRNAs
that are generated by Dicer cannot and rely instead upon
siRISC assembly pathways (Figure 2).
Figure 3. Mechanisms of siRNA Silencing 26
siRNAs Can Induce Heterochromatin
siRNAs are not restricted to posttranscriptional modes of
repression. In 2002, siRNAs were shown to induce
heterochromatin formation in S. pombe, consistent with
earlier reports of transcriptional gene silencing (TGS) in
MicroRNAs in the plant and animal (Figure 4)
Figure 4. Biogenesis of miRNAs and Assembly into miRISC in Plants
and Animals 32
in humans, C. elegans, and Drosophila indicates
Posttranscriptional Repression by miRNAs
The miRNA acts as an adaptor (Figure 5)
The degree of miRNA-mRNA complementarity has been
considered a key determinant of the regulatory mechanism.
Figure 5. Possible Mechanisms of miRISC-Mediated Repression 35
dsRNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the dsRNA and the target gene/mRNA is
Targeted mRNA is lost (degraded) after RNAi
The effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA