L6, Euk. Regulatory RNAs Flashcards
How do regulatory RNAs usually operate?
- They are processed from their original transcript to yield the functional molecule
- They typically employ base pairing with their RNA and DNA targets, often employing interactions with other components
Give the 4 types of eukaryotic regulatory RNA:
- siRNA (small interfering)
- miRNA (micro)
- piRNA (piwi-interacting)
- lncRNA (long non-coding)
- All but lncRNAs are classed as sRNAs (involving RNA interference)
What is RNA interference? (classic pathway first identified in C.elegans)
- dsRNA broken down by dicer
- dsRNAs of 20-25nts produced (guide and passenger)
- Guide is loaded onto Argonaute -> confers specificity for binding to target RNA
- RISC complex forms at target RNA -> carries out silencing to regulate gene expression in various ways
In what 5 ways does RISC bind influence gene expression:
- Inhibiting transcription
- Forming heterochromatin
- Inhibiting translation
- RNA degradation
- DNA degradation
Where are miRNAs, sirRNAs and piRNAs derived?
- miRNAs: endogenous primary transcripts i.e. genes; capped and polydenylated
- siRNAs: dsRNAs from various sources including viral, non-coding regions etc)
- piRNAs: processed from long single stranded precursor transcripts e.g. rasiRNA
What does rasiRNA stand for and where are they derived from?
- repeat-associated small interfering RNAs
- Derived from repetitive regions of the genome
- Particularly important during development (protective role)
Drosha vs Dicer structure:
- Drosha: 2 catalytic domains and RNA binding domain
- Dicer: same as Drosha but with PAZ domain which anchors the 3’ end
How is eukaryotic miRNA processed in the nucleus?
- pri-miRNA folds into stem loop structure
- 5’ and 3’ extensions are removed from pri-miRNA by microprocessor complex (Drosha and DGCR8)
- Hairpin is produced (with 3’OH and 5’monophosphatem which is exported from the nucleus
- In the cytoplasm, the PAZ domain of Dicer binds the 3’ end -> cleaves pre-miRNA to produce miRNA:miRNA
- These transcripts are not usually fully complementary
How is eukaryotic siRNA processed?
- Does not occur in nucleus!
- siRNAs are derived from dsRNAs from various sources in the cytoplasm
- Dicer and TRBP2 sequentially chop to produce 20-25nt siR:siR molecules
- Transcripts are usually fully complementary
What is the key role of siRNAs?
- Cellular defense against exogenous RNAs
2 key similarities between miRNAs and siRNAs:
- Both have 3’OH and 5’monophosphates
- Both can undergo PTMs e.g. methylation
4 domains of argonaute proteins with functions:
- PAZ; binds 3’ end of bound RNA
- Mid; interacts with 5’ end of RNA
- PIWI; related to RNase H, interacts with whole RNA
- (N domain)
- PAZ, Mid and PIWI together orient the bound guide RNA to facilitate its scanning of cellular molecules
What is the name of deciding between guide and passenger strand of si/miR? What is the function of the guide strand?
- Sorting
- Involved removing the passenger after initial loading
- The remaining guide strand is involved in silencing target RNA
What is the difference between guide strand loading in si/miR vs single stranded piRNA?
- piRNAs are loaded directly onto PIWi proteins as they are generated
- No sorting is required
How do miRNAs typically influence their target?
- Usually imperfect match with target
- Translation inhibited and mRNA degraded
How do miRISCs vs siRISCs bind? Affects of binding?
- miRISC usually pairs initially via a 2-8nt seed sequence (since the match is imperfect); 3’ end remains tightly bound to PAZ domain -> represses transcription of target
- siRISC is a perfect match, binds along full length (at ORF of target). This releases the PAZ domain and induces a conformational change that activates the catalytic activity of the PIWI domain -> degradation of target
lin14 regulation in C.elegans:
- Regulated by lin4 miRNA
- Binds to 3’UTR of lin14 mRNA -> repression
How are miRNAs involved in cancer?
- miRNAs help to regulate cell signalling pathways and cell proliferation
- Able to repress TSGs and oncogenes
- Global dysregulation of miRNAs observed in many cancers -> potential therapeutics
By what different mechanisms can miRNA dysregulation cause cancer?
- Overexpression of TSG targeting miRNAs -> downregulation of TSG
- Loss of oncogene targeting miRNAs -> upregulation of oncogenes
- Loss of DNAmethyltransferase -> downregulation of TSG
- Loss of chromatin silencer -> downregulation of TSG
How are piRNAs generated? When are they particularly useful?
- Generated from long transcripts, usually from repeat associated regions using a ping pong mechanism
- Produced in germ cells in most animals; rigorously prevent inappropriate transcription of viral elements during development
How are piRNAs processed?
- Transcripts are first processed by the PIWI domain of Piwi protein to yield primary piRNAs (pre-pre-piRNA) -> direct secondary processing and production of secondary piRNAs that direct further cleavage upon different AGO complex (evolutionarily conserved domains)
- Called ping pong cycle, with initiator and responder piRNAs
Role of ping-pong mechanism, role of trailing piRNAs:
- Amplifies initial piRNA
- Phased pathway chops up remainder -> new inititiator piRNAs (‘trailing piRNAs’) -> confers diversity of sequence
3 key roles of piRNAs:
- Transposon silencing (particularly in germline)
- Viral defence (in somatic tissues; secondary to defence by siRNAs, generally speaking)
- Regulation of gene expression (e.g. meiotic mRNA regulation by pachytene piRNAs in male germ cells)
lncRNAs: Features (x3)
- Over 200 nt
- no ORF
- arise from most of eukaryotic genome
SER3 (in yeast) regulation by lncRNA:
- SRG1 (lncRNA) blocks transcription of SER3
fbp1 regulation by lncRNAs in yeast:
- lncRNAs transcribed upstream of fbp1 -> opens up chromatin
- This occurs under glucose starvation; inhibition is released by action of lncRNAs
