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: 1.The ribosomes cannot gain access to nucleotides in mRNA 2.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 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: 1.Perhaps by binding to complementary sequences on DNA 2.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 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… 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.

 Why RNA triggers and DNA does not?

 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

Any questions???