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
