Topic 12 Flashcards
Small interfering RNAs (siRNAs)
Small non-coding RNAs of ~21-23 nucleotides produced from longer dsRNA precursors
MicroRNAs (miRNAs)
Short single-stranded, non-coding RNAs of ~19-25 nucleotides produced from hairpin RNA precursors (stemloop structure generated as a ssRNA that was folded back into a hairpin)
Dicer
An RNase-III like enzyme that recognizes and digests longer dsRNA to form siRNAs or the stem-loop structures to form miRNAs
Microprocessor complex
Consists of Drosha (an RNase-III like enzyme similar to Dicer) and Pasha/DGCR8 responsible for initiating the biogenesis of miRNA pri-miRNA to form pre-miRNA
RNase III enzymes are specific for…
dsRNA and the resulting product has 2 nucleotides overhanging on the 3’ end
Both Dicer and Drosha recognize and cleave pri-miRNA on the basis of…
The pri-miRNA structure rather than its specific sequence
RNA-induced silencing complex (RISC)
A multi-protein complex that includes a guide RNA, which is the active form of the regulatory RNA
Argonaute protein
The catalytic (i.e. slicer) subunit of RISC that carries out the mRNA cleavage
Describe the steps of RNAi silencing (5 steps)
- pri-miRNAs are processed in nucleus by microprocessor complex (Drosha+Pasha) to form pre-miRNA
- Pre-miRNAs or dsRNA are processed by Dicer in cytoplasm to form mature miRNAs or siRNAs
- Incorporation of ds-regulatory RNA into RISC
- De-naturation of dsRNA into a single-stranded guide RNA and a passenger RNA (degraded complement of guide RNA)
- The guide RNA base pairs with the target mRNA to promote silencing of gene expression by triggering the argonaut in the RISC to cleave mRNA
What are the 3 ways that RNAi can silence gene expression?
- Through mRNA degradation
- Translational repression
- Transcriptionally silence genes by directing chromatin modification
siRNA (does/does not) fully complement the target mRNA. miRNA (does/does not) fully complement the target mRNA.
Does, does not
Mature miRNA is generated from which reactions?
pri-miRNA by 2 RNA cleavage reactions:
- pri-miRNA (hairpin structure) -> first cleavage by microprocessor complex -> pre-miRNA (stem loop)
- pre-miRNA -> second cleavage by Dicer -> mature miRNA
Do introns or exons encode miRNAs?
Both
Seed residues
The sequence between bases 2 and 9 of miRNA and are important for determining the base pairing between miRNA and its target RNA
What techniques can be used to detect the presence of an miRNA?
Northern blotting and qPCR
Describe the pri-miRNA structure (4 regions)
- Basal segments don’t base pair: important for microprocessor complex (Pasha/Drosha) recognition
- Lower stem region
- Mature miRNA region/upper stem that isn’t perfectly complementary, seed residue contained within this region
- ssRNA-dsRNA junction (where ssRNA meets dsRNA) contained at start of this region, which determines Drosha cleavage specificity
- 2 helical turns - Terminal loop
- Variable in size
- Sequence is not important for processing
True or false: the miRNA sequence is not important for processing
True
Describe how the microprocessor complex processes pri-miRNA
- Drosha makes 2 cleavages at the ssRNA-dsRNA junction
- 3 fragments are generated
- The F2 pre-miRNA has 2 nucleotides overhanging on its 3’ end, which is important for Dicer recognition in the cytoplasm
What are the 3 modules of Dicer?
- 2 RNase III domains
- PAZ (Piwi, Argonaute and Zwille) domain: dsRNA-binding domain; anchors the 3’ end of the dsRNA substrate and positions it to the active site at ~22 nt away
What are the 3 steps of formation of guide strand RNA and RISC activation?
- The short (~21-25 nt) double-stranded RNA generated by Dicer is incorporated into RISC
- The dsRNA is de-natured into a guide strand and a passenger strand
- The passenger strand is removed from the complex
- The resulting RISC is called the mature RISK
siRNA and miRNA usually bind to which region of the mRNA?
3’UTR
Describe the structure of the Argonaute (2 domains)
- A PAZ domain: specifically recognizes the 3’ end of the guide RNA
- An RNase domain (RNase H activity)
The RNase active site of the argonaute is positioned…
To cleave in the middle of the paired region between small RNA and mRNA
Mature RISC can inhibit translation, but the mechanism remains to be elucidated. What are some potential ways that researchers think this is done? (2)
- miRNA may sequester the target mRNA to processing bodies, where translation is repressed in the cytoplasm
- miRNA binding can also destabilize the polyA tail of the targeting mRNA
Describe how genes are silenced by chromatin modification in S.pombe (4)
- Each centromere in S.pombe has a central unique sequence flanked by common repeats that are important for heterochromatin formation and transcriptional silencing
- The repeats are transcribed from both strands by RNA Pol II to form dsRNA, which is then processed by the RNAi machinery to generate siRNAs
- siRNAs direct RITS complex to the Pol II-tethered transcripts
- The complex further recruits factors (Swi6 and Clr4) to modify nucleosomes by adding the H3K9 silencing markers
Describe RNAi used for X-chromosome inactivation
Female mammals: XX
Male mammals: XY
- females need to inactivate one of the 2 X chromosomes for dosage compensation to ensure both sexes express comparable amounts of X chromosome gene products.
- Xist RNA is encoded in the Xic on the X chromosome
- Xist RNA recruits other factors to modify and condense X chromosome, which leads to X chromosome inactivation
Describe RNAi as a research tool
RNAi is a powerful tool for manipulating gene expression through knockdowns
- C. elegans eat plasmids with shRNA inserts
What are small RNAs in prokaryotes? Describe their structure
- 80-110 nucleotides long
- Encoded by small genes
- Mostly work by direct base pairing with the target mRNAs to regulate gene expression
- Hfq, an RNA chaperone, facilitates binding of sRNA to its target mRNA
What do riboswitches regulate?
Regulate gene expression at transcription or translational levels by controlling metabolic operons and attenuation in biosynthetic operons through their response to changes in the concentration of small molecules
What are the two components of cis-regulator RNAs (riboswitches)?
- Aptamer: binds to the small molecule ligand and undergoes conformational changes
- Expression platform: undergoes secondary structural changes upon aptamer-controlling metabolite binding (after comformational change)
Describe regulation of transcription termination by riboswitch using SAM
SAM: S-adenosyl mehionine is a metabolite
- In bacteria, SAM is bound to the SAM sensing riboswitch, which in turn regulates genes involved in methionine or cysteine biosynthesis
- After SAM binds aptamer, regions 1-2 and 3-4 start base pairing instead of 2-3 in the aptamer, and the 3-4 stem loop acts as a transcriptional terminator
Describe regulation of translation initiation by riboswitch using SAM
When SAM binds, conformational change in aptamer moves ribosome binding site (RBS) on region 4, making RBS inaccessible to the ribosome and inhibiting translation
sRNA is said to be a…
Trans-acting regulatory RNA
- It can activate and repress translation
