How can we introduce dsRNA to impact a gene?
Examples of flies, plants and mice
- dsRNA allows introduction of specific transgenes to make loop (folds back on itself) that correspond to specific targets
- flies: have red eyes due to a white gene, if we eliminate by introducing snap back, gives white eyes
- plants: introduction of transgene gives mRNA that causes multiple stem cells
- mice: introduction of siRNAs allows us to make a phenocopy of Tay-Sachs. disease eliminates siRNA early - do the same things
This process is essentially the introduction of dsRNA or siRNA to recap the phenotype of certain diseases
Explain the DICER-RISC system
- DICER chops up dsmicroRNAs into small fragments
- RISC (RNA inducing silencing complex) has a protein calles Argonaute which binds to these fragments
- Helicase unwinds the fragments with ATP hydrolysis, creating ssRNA
- ssRNA targets an mRNA to be degraded, binds to it
- this new complex is now cleaved and degraded
What is RNAi vs microRNA
- microRNA will be destabilized for inhibition of translation, interacts with RISC and is no 100% complementary
- siRNA/RNAi is 100% complementary, and will be degraded - it is sequence specific suppression
How is chromatin involved in cell division?
Chromatin MUST be silenced within the centromere for accurate cell division to take place
What does H3K9me3 do?
It is a repressor of lineage innapropriate genes
Make by dsRNA nucleate complex
Describe piRNA and PIWI
piRNA is transcribed from a DNA cluster made up of integrated disabled transposable elements
- We use argonaute PIWI to cleave their RNA
They regulate mRNA stability and enhance protein synthesis
What is the role of ncRNA?
Fine tunes physiological processes as organisms develop
novel cellular RNA
What are two strategies used by viruses on RNA during RISC process?
- Make long RNA with repeating units, binding up all micromRNA
- Make circular RNA that soak up the micromRNA important for defense
What is a Barr body?
Females have barr bodies - inactivated X chromosome
Can see it in calico cats- different coat colour shows deactivation of X chromosome
Explain the XIST gene
XIST locus encodes lncRNA (long non-coding RNA)
- it binds to X chromosome region and extinguishes its gene expression
- inactive X chromosome is due to XIST expression or not
- it recruits complexes that modify chromatin, condensing chromosome, making it inaccessible to transcription factors
Explain XIST vs TSIX
- XIST silences X chromosome
- TSIX antagonizes XIST expression
