What makes siRNA in the RITS pathway?
dicer
What does the RITS pathway lead to?
transcriptional suppression
What does the complex recruit in the RITS pathway?
histone methylases
What are the steps of the RITS pathway?
- Dicer recognizes ds RNA
- Dicer cuts the RNA into ds siRNAs
- Argonaute and proteins unwind (helicase) the ds to ss RNA, keeping one strand as a guid and letting the other (passenger) be degraded
- The RITS complex and guide RNA are release in the cell and try to match with the 5’ end of a new RNA
- If there is a match the proteins recruit chromatin closing proteins (histone methylases and DNA methylases) to stop transcription
Why do we have RNA interference (RNAi) mechanisms?
Long ds RNA in our cells can be a signal of the presence of some invading viruses or excess transposing activity
Both RISC & RITS pathways use what 2 things?
argonaute complex & a dicer
What is the purpose of the RISC pathway?
leads to immediate cleaveage and degrades RNA that matches gRNA
What are the steps of the RISC pathway?
- Dicer (endonuclease) produces siRNAs of about 22 bp with overhangs by cleaving both strands of long dsRNA.
- Argonaute (endonuclease + helicase) and other RISC proteins open the ds siRNAs, take one strand to become “guide RNA”. The other strand gets degraded (degraded strand = “passenger strand”).
- If there is exact W/C bp on the guide RNA with another ss RNA sequence leads to cleavage of that RNA.
- An Endonuclease from the Argonaute complex with cut up the rest of the RNA.
What is the purpose of the RITS pathway?
Stops transcription when gRNA W/C bp with 5’ end of actively synthesizes RNA
What do micro-RNAs look like?
hair pins
miRNAs are produced to W/C bp with mRNA to inhibit what?
translation
How do miRNAs prevent translation?
interacting with the 3’UTR of mRNAs
Why do miRNAs have a 5’ cap & a 3’ poly-a-tail?
because they are made by RNA polymerase 2
What are the steps for miRNA?
- miRNA is produced in a hairpin structure
- In the nucleus, DICER cuts the cap and tail from the miRNA and the modified miRNA leaves the nucleus
- In the cytosine, DICER cleaves the structure again removing the hairpin shape
- RISC uses its helicase and endonuclease function to create a guid and passenger strand of RNA
- outcomes
* If perfect match, leads to degradation immediately of the mRNA so no protein creation
* If not perfect match, leads to translational repression and EVENTUAL degradation
miRNAs can be transcribed from what regions?
intronic
What is the Piwi complex?
- same as RISC but in germ cells
- piRNAs are used to regulate transposisition in 2 major ways:
Transposon degradation &
Transposon transcriptional silencing
What is the piwi complex specifically targeting?
Targeting RNAs that come off pi transposons
What are the steps of the piwi complex?
- “Detection”: The piRNA cluster is found of the chromosome. RNA polymerase II will transcribe piRNAs from the cluster
- “Amplification”: as the known Transposon sequence RNAs are being produced, loaded piwi proteins (a piwi protein with a piRNA being used as a gRNA/guid strand) will bind to these Transposon transcript to make more proteins
- “Repression”: the loaded piwi proteins can regulate transposons in two major ways
- Repressing transcription using DNMT (by methylating the DNA)
- Repressing transcription by histone modifications (chromatin condensation)
When can infertility result in piRNA?
if piRNA proteins ARE NOT degraded
What defines germ cell stem cells?
presence of ddx4 (RNA helicase and marker for stem cells to develop egg cells )
What is the mouse vasa homolog (mvh)?
mouse version of ddx4
What is the total RNA binding domain?
SR & deadbox
What is RNAi?
as a way to destroy unwanted, especially viral RNAs
What are the effects of non-coding RNAs?
- RNA degradation
- inhibit translation
- gene/transcription silencing
- miRNA inhibit transLATION
- endo siRNA inhibits transCRIPTION
