Long non-coding RNA (LncRNA)s are larger than 200 nucleotides in length and are pervasively expressed across the genome
Some lncRNAs have been implicated in transcriptional and post-transcriptional gene regulation, including control of RNA and protein synthesis, RNA maturation, RNA transport, and transcriptional gene silencing via epigenetic regulation and chromatin remodeling
How can we classify lncRNAs?
Based on their mechanism of action:
Signal
Decoy
Guide
Scaffold
CircRNA
Circular RNA
CircRNAs are formed from pre-mRNAs via backsplicing of exons, introns, or their combinations
circRNAs are resistant to 5' and 3' exonucleases
T/F
True
circRNAs have no free 5’ and 3’ ends and are resistant to 5’ and 3’ exonucleases
Many circRNA are artefacts of splicing, sequencing, or annotation
When was the first demonstration of RNAi recorded in plants?
1990
"introduction of chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans"Expression of multiple copies of pigment transgene (chalcone synthase) resulted in white or variegated flowers (Napoli C., et al.)
In Fire and Mello's experimental design investigating RNAi in C.elegans, which form of RNA was most effective at silencing the target gene:
A) dsRNA
dsRNA was orders of magnitude more effective than ssRNA at silencing the target gene (a few copies per cell were sufficient!)
What is a summary of observations by Fire and Mello, following their experiments investigating RNAi in C.elegans?
Silencing was triggered by dsRNA
Silencing was specific to the mRNA homologous to dsRNA
dsRNA had to correspond to mature mRNA sequence, not promoter or intron sequences
Target mRNA disappeared suggesting degradation
Silencing effects “crossed cellular boundaries”: injection into adult worm’s head and tail caused silencing in the progeny
dsRNA could interfere with the expression of multiple genes that share homology with the dsRNA
What cleaves target mRNA?
RISC
describe the functional domains in Dicer
Helicase
PAZ domain
2 RNAse-III domains
dsRNA binding domain
What does the protein Dicer do?
Cleaves dsRNA into 19-25nt siRNAs: Leaves 3’ overhangs and 5’ phosphate groups
Cytoplasmic RNAse-III enzyme
Multiple Dicer genes in Drosophilia and plants, but only one in mammals
What does the PAZ domain of Ago bind to?
PAZ domain of Ago binds the characteristic two-base 3' overhangs of siRNAs
What is the role of Ago gene?
Argonaute (Ago)
Ago plus siRNA constitute the minimal RISC complex
What confers the 'homology seeking' activity to RISC in the Argonaute gene?
Homology seeking (aka sequence specificity) is dictated by the siRNA or miRNA, not by AGO itself
shRNA
short hairpin RNA
can trigger RNAi
What is a liposome?
Liposomes are artificial vesicles that are positively charged and can form complexes with negatively charged nucleic acids.
These complexes are assumed to have net positive charges and allow cells to take up the plasmids easily.
Cells uptake the contents of the liposomes either by membrane fusion or via endocytosis.
miRNA vs siRNA
Similar biogenesis and size (20-25nt)
siRNA
miRNA
Mostly of exogenous origin
encoded by endogenous genes
made out of dsRNA precursors
made out of hairpin precursors
may be target-specific
recognize multiple targets
Full complementarity with the target mRNA
partial complementarity with the target mRNA
siRNA vs miRNA (image):
Where are most miRNA genes located?
Most miRNA genes are located in intergenic regions or in an antisense strand of other genes and are transcribed by RNA Pol II as larger precursors, pri-miRNA
Pri-miRNA
Primary miRNAs
Pri-miRNAs consist of one or more hairpin structure (each made of a stem and a terminal loop)
Pri-miRNA transcripts are capped and polyadenylated, some have introns
