Regulation of Gene Expression by RNAs
A Control Experiment Showed that Double-stranded RNA is a Trigger for mRNA Degradation
In situ hybridization experiments with C. elegans eggs.
We are looking for the mRNA for Mex-3
Mex-3 Antisense RNA Treatment
Antisense RNA would be complementary to the Mex-3 mRNA to see if it could get degradation of the Mex-3 mRNA.
Mex-3 Sense RNA treatment
Sense RNA is the same with the sequence of Mex-3 mRNA.
The sense RNA had some dsDNA contamination.
Double stranded RNA induced phenomenon called RNA Interference.
RNA Interference/RNA i
How does RNAi work?
- Dicer cleaves the dsRNA(has RNase III activity) into 18-20 nt double stranded pieces.
- small dsRNA unwound by an RNA helicase
- ssRNA loaded into RISC (RNA-induced silencing complex). RISC will take the single stranded RNAs that are complexed with other proteins. And they will help find complementary mRNA sequences on mRNAs.
- ssRNA binds to complementary mRNA
- mRNA 'sliced' by Argonaute protein in RISC complex.
The Argonaute Proteins are Responsibel for mRNA Cleavage
RNAi can be Amplified and is Often Systemic
C.elegans has really strong RNAi mechanisms.
Part of this is due to the presence of RNA-directed RNA polymerase, which increases the extent of small interfering RNAs(siRNAs )
Micro RNAs(miRNAs) are Endogenous Genes that Regulate Gene Expression
mRNAs are repressed but not degraded.
miRNAs are Required for Proper Development and Maintenance of Many Tissues
CircRNAs are Common and Have Important Functions in Cells
Functions: regulate gene expression. Can 'sponge' miRNAs or RNA binding proteins. Can promote transcription.
Many Classes of Small RNAs Have Been Discovered
What are long, non-coding RNAs(IncRNAs)?
- RNAs longer than 200 nucleotides that do not have protein-coding potential
- They perform many regulatory functions in cells
- Implicated in cancer progression
Summary of how IncRNAs can Exert Their Effects
a. bind to protein and prevent it from being able to associate with the double stranded DNA.
b. act as RNA scaffolds to bring together different protein subunits.
c. act as guides to interact with double strand DNA or other protein to bring those to DNA.
d. Act as enhancers or interact with double stranded DNA far upstream of genes. Bringing together as a scaffold. Other proteins that bind to promoter sequences. Through this looping, they can induce transcription of initiation.
IncRNAs are often Associated with Chromatin Modifier Enzymes that Regulate Gene Expression
Lnc non-coding RNA binding to transcription cofactor and chromatin modifiers. By doing this, it allows for the promotion of Gene Expression.
In final journal club paper, with chromosome 11, it shows cheomosome 11 is going to be important for bringing a particular transcriptional protein to the promoter of the topoisomerase to a gene in eukaryotes.
May look carefully at the protein the rams 11 lncRNA is interacting with. To see if it might have additional functions. And you might want to think about other experiments that you could do to potentially query whether or not these functions are important.