siRNA and Epigenetic Asma Siddique Saloom Aslam Syeda Zainab Ali
Topics under discussion • Brief History • Intro to different terms • What is RNAi? • What is siRNA? • siRNA formation • Difference between miRNA and SiRNA • siRNA design • Therapies • Challenges • Clinical trials
History • SiRNA was first discovered by David Baulcombe’s group as part of post-transcriptional gene silencing in plants, in 1993.
Different terms • RNAi • siRNA • shRNA • MiRNA • RISC… DICER, ARGONAUTE FAMILY PROTEINS and OTHER PROTEINS • Off- target effects
What is RNA interference? • Gene silencing mechanism...siRNA and miRNA • Known as the RNA interference machinery. Once it finds a double-stranded RNA (Dicer), separates the two molecule, cuts it up. • Way to silence genes by preventing the formation of the proteins that they code for.
Transitive RNAi • Organisms have RNA dependant RNA polymerase that uses the mRNA targeted by the initial anti-sense SIRNA as a template for the synthesis of more siRNA. • These secondary siRNA also target other parts of mRNA.
When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked: • The ribosomes cannot gain access to nucleotides in mRNA • Duplex RNA is quickly degraded by ribonucleases
Double stranded RNA corresponding to a particular gene is a powerful suppressant of that gene. • The suppressive effect of anti sense RNA probably depends on its ability to form dsRNA.
siRNA • siRNA known as short\small interfering RNA, are a class of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes. It has 2-nt overhangs on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group. • They are produced as part of the RNA interference (RNAi) pathway by the enzyme Dicer. • They can also be exogenously (artificially) introduced by investigators to bring about the knockdown of a particular gene.
Sources of siRNA • Plant cells make these from the double stranded RNA of invading viruses. • Scientists make these as agents to turn off the expression of specific genes.
SiRNA formation • Delivery of trigger dsRNA • Generation of siRNA pool • Capture, unwinding of SiRNA by RISC • Binding of SiRNA associated RISC with target mRNA… ATP dependant • Destruction of target mRNA
SiRNA can also inhibit the transcription of genes: • Perhaps by binding to complementary sequences on DNA • Perhaps by binding to the nascent RNA transcript as it is formed.
How these SiRNAs synthesized in the cytosol –gain access to the DNA in the nucleus is unknown.
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. • They act by either destroying or inhibiting translation of several mRNA (by binding to a region of complimentary sequences in the 3’UTR of mRNA)
Studies have shown that miRNAs play a role in the most critical biological events including development, proliferation, differentiation, cell fate determination, apoptosis, signal transduction, organ development, hematopoietic lineage differntiation, host viral interactions and carcinogenesis.
Effective SiRNA design? • Select the target region from the open reading frame of a given DNA sequence…50-100 nt down stream of the start codon. • Search for sequences 5’AA(N19)UU, in the mRNA sequence and choose those with approx 50% GC content. • BLAST search • Strand incorporation depends upon weaker base pairing…more AT content more incorporation.
Therapies • Synthetic siRNA molecules that bind to gene promoters can repress transcription of that gene. Repression is mediated by methylation of the DNA in the promoter ; methylation of histones in the vicinity. • Rnai can use as a weapon to counter infections by RNA viruses by destroying their mRNA’s. • Screening genes for their effect on drug sensitivity.
More tightly packed • More stable • RNA is easily hydrolysed
Challenges of RNAi • Finding a vector or delivery system • At what age, a patient should receive treatment • RNAi therapy is long term or only temporary? • Long dsRNA fragments reduce gene expression in mammals
Clinical trials underway • “wet” macular degeneration (targeting VEGF which encodes vascular endothelial growth factors) • AIDS (targeting an exon used by the HIV envelope protein) • Hepatitis B (targeting four different sequences in the viral genome) • Some cancers