Topic 12 Regulatory RNAs Flashcards
What are regulatory rna
What is RNAi
non coding RNA, don’t encode for proteins
RNA interference (RNAi): it’s a category of regulatory RNA that silences mRNA expression
They are short 19-25 nt and made from longer double stranded RNA
What are the categories of RNAi machinery
When we’re they discovered
Small interfering rna (siRNA):
when mature they are small non coding rna 21-23 nt long that are made from longer dsRNA precursors
Double stranded structure
MicroRNA (miRNA):
when mature they are short non coding RNA 19-25 nt long made from longer stem loop rna precursors
Single stranded stem loop structure
Pi-RNA: found only in the germ-line, regulates transgenerational gene expression by suppressing the function of transposable elements
MiRNA was discovered earlier (1993) than siRNA in c. Elegans
But they thought it was only in c Elegans, now know miRNA is in humans as well
What are the key components that make RNAi machinery (non coding rna)
Dicer
Microprocessor complex
RNA induced silencing complex
Argonaute protien
What is dicer
An RNase-III like enzyme that processes long dsRNA to make siRNA or stem loop structures to make miRNA
What is the microprocessor complex
Made of two components as a heterodimer:
Drosha: RNase-III like enzyme with RNase activity (similar to dicer)
Pasha/DGCR8: recognition of the proper miRNA to undergo processing
cleaves primary miRNA (pri-miRNA) to make miRNA precursors (pre-miRNA) which can be turned into mature miRNA
THIS COMPLEX ONLY MAKES MIRNA NOT SIRNA
Whag is the formation of non coding RNA like miRNA and siRNA called
Biogenesis
What is RISC
What is the argonaute protien
A multi protien complex that has the guide rna which is the active from of miRNA or siRNA
It brings these regulatory rna to the target
mrna to regulate the expression
Argonaute protien: a key slicer component of the RISC complex which cleaves the target mRNA leading to gene silencing (since no more mRNA)
Slode 5 note
Idk
Decribe how siRNA undergoes biogenesis
1.long dsRNA introduced to the cells through foreign particles (viruses)
- Dicer chops the long dsRNA to short dsRNA (22nt), this dsRNA is loaded into the RISC complex
- When on RISC complex, the two strands of dsRNA melt and becomes two separate strands
- One strand is the passenger strand: gets degraded
other strand called guide strand (siRNA): stays on the risc complex to guide it to the target mRNA
- the siRNA guide strand compliments the sequence of the target mRNA which activates the Argonaute to degrade the target mRNA
- No gene expression since mRNA degraded
Decribe how miRNA undergoes biogenesis
MicroRNA need to undergo multiple cleavages
- Primary miRNA (pri-miRNA):
200 nt in length folded into single stranded stem loop structure
This is processed in the nucleus by the microprocessor complex to make precursor mi RNA (pre miRNA (70 nt))
All in nucleus so far
- Pre miRNA is sent to the cytoplasm through the nuclear pore complex and is processed by Dicer into 22 nt mature miRNA in the cytoplasm
- The 22 nt miRNA binds to miRNA risc in the cytoplasm
- The stem loop structure disscosistes and becomes passenger strand and guide strand
- The miRNA is not 100% compliment to the targeting RNA
Whag is the functional difference between miRNA and siRNA
SiRNA is 100% complaints to its target RNA, miRNA isn’t
SiRNA and miRNA have their own separate risc complexes that they bind to
How does the RNAi silencing guide rna promote gene expression silencing
- MRNA degradation
- Translational repression
- Return to nucleus and silence genes by directing chromatin modifications to make heterochromatin (transcription is repressed)
Describe the structure of miRNA
Pri miRNA transcribed as a single rna transcript
Made from pri- miRNA by 2 rna cleavage reactions
Primirna is hairpin structure, cleaved by microprocessor complex, make pre miRNA as a stem loop structure in nucleus
Premirna cleaved by Dicer to make mature miRNA in cytoplasm
How can miRNA be found by bioinformatics
Can look at rna sequence and see if they have seuwences that fold back to form a stem loop structure
What sequences encode pre-miRNA
From the coding, non coding, 3’ UTR, or intron sequence
Or in combo of all of those
they are everywhere to regulate a variety of things
What are seed residues
Sequence between bases 2 and 9 of miRNA that determine the base pairing between miRNA and the target RNA
But it’s not perfect base paring which is why miRNA has less complimentarity to the target mRNA
What techniques can be used to detect prescience of non coding regulatory rna like siRNA and miRNA
Northern blot and qPCR
Describe the structure of pri-miRNA
How many turn of helix is mature miRNA
Expressed as a single stranded loop structure
Basal segments: At the 3’ and 5’ end of the structure, there is no base pairing (no loops) which is important for the microprocessor to recognize the pri-miRNA
Lower stem ss and ds rna junction: There are bulges between the base paired region that are recognized by drosha for cleavage
Upper stem Mature miRNA: 22 nt region in the priMIRNA that has perfect base paring , this is the mature 22 nt miRNA when fully processed
Is around 2 turns of the dna helix (10.5nt x 2)
Seed sequence: after the +1 site of the mature miRNA sequence (between nt 2-9)
terminal stem loop: variable in size and cleaved off by Dicer in the cytoplasm , the sequence of this isn’t important for processing
Describe how the microprocessor complex cleaves pri-MIRNA to pre miRNA
Happens in nucleus
- Pasha (recognition subunit) and drosha (catalytic subunit) amale ip the microprocessor complex and recognize the pri-miRNA
- Drosha makes two cleavages at the ss and ds rna junction
- Three fragments are made, fragment 2 is the pre-miRNA which has a 3’ overhang of 2 nt
So now 11 BP and 22bp split
- The pre-miRNA 3’ overhang is important for Dicer recognition in the cytoplasm
Describe the structure of Dicer
Has 3 domains
2 RNase III domains:
do the cleavage of the stem loop of premirna
PAZ domain (Piwi, Argonaute, Zwille):
dsRNA binding domain that helps hold the pre-miRNA structure
Binds to the 3’ overhang of the premi rna and positions the RNase III domains to the cleavage site 22 nt away
What is similar in Drosha and Dicer
They are both RNase III enzymes that cleave miRNA
How does the Dicer cleave the pre miRNA
The premirna is sent to cytoplasm
The cleavage occurs and the pre miRNA becomes 22 nt long mature mi rna
How does the guide miRNA get formed and risc get activated in miRNA biogenesis
Guide rna is the active singles stranded from of the regulatory rna (in this case miRNA)
1 After Dicer cleavage the 22 nt miRNA is double stranded RNA
- DsRNA goes to risc (now called miRISC)
dsRNA is denatured into a single guide strand and passenger strand
- Passenger strand is degraded, guide strand recognizes the target RNA and guides risc to it
- RISC with the single guide miRNA is the mature risc
- The guide RNA recognizes and base pairs with target mRNA
What is the structure of the Argonaute
Argonaute within the risc complex does the mRNA slicing
has the PAZ domain that recognizes the 3’ overhang end of the guide rna
the RNase H domain (to degrade using RNase H activity)
Different from Drosha and Dicer because they have RNase III activity
Explain how the RISC complex cleaves the target mRNA using miRNA
- The guide rna brings the miRISC to the target mRNA
- Guide stand non perfectly compliments the 3’ UTR mRNA sequence because of the seed sequence so we see bulges
- This binding of the guide strand sends signal to the Argonaute to degrade the target mRNA through its RNase H activity
- Since rna is chopped, gene silence no protien made, low gene expression
Explain how the RISC complex cleaves the target mRNA using siRNA
The cleaved dS siRNA by Dicer is loaded onto risc
This makes siRISC, the two strand are melted into passenger and guide
Guide send siRISC to target mRNA, binds with perfect complimentary to 3’ UTR of mRNA
Argonaute cleaves the mRNA
How does the guide strand bind to the RISC compelx
What types of cleavage does the microprocessor complex do
Bind to the argonoir because the Paz domain binds to the 3’ end of the rna
Sticky end not blunt because it makes a 3’ overhang
Overall in function how is siRNA diff from mi rna
SiRNA is more precise for the mRNA target, miRNA has more ambiguity because less complimentarity so it leads to off target effects
What other ways can risc silence genes
Inhibits translation
- by the miRNA sequestering the target mRNA to processing bodies. These processing bodies (P-bodies/p-granules) are in the cytoplasm and repress translation
- The binding of miRNA destabilizes the poly-A tail of the target mRNA. The poly A tail recruits important translation initiation factors so now translation repressed
- Introduce chromatin modifications to make heterchromatin, transcription gene silencing because packed too tightly
Explain how gene silencing by chromatin modification can happen using regulatory rna
In the dna of S. Pombe, there is an open region of repeats that allows heterochromatin formation and gene silencing
This both dna strands in this region is transcribed by rna pol II into dsRNA which is then processed into siRNA
The dS siRNA binds to the RITS complex (rna induced transcriptional silencing complex)
It binds to the mRNA target that is being transcribed by pol II and recruits chromatin modifiers Swi6 and Clr4 to the dna
These modifiers methylate the H3K9 histones and make heterochromatin
What are the biological functions of RNAi
- Helps against viral infection in plants and humans
- Helps in x chromsome inactivation in the form of xist RNA at the Xic (x inactivation centre) . This rna recurits factors to condense the x chromsome whcih causes inactivation
Describe the first phenomena of RNAi
Crossed two dark flower in hope of making a black flower
Found that the product becomes white instead
The genes they crossed together silence each others pigmentation
How can RNAi be used as a research tool
To study the function of a gene
Knock down: reduce the expression of that gene then see the change in genotype, not completely knock out by the RNAi
Ex.
Have plasmid and insert gene of interest into it, inside the plasmid the gene is flanked by two promoter regions which are bound by T7 polymerase
Introduce the plasmid into E. coli
The T7 polymerase transcribes dsRNA of the gene of interest (transcribes both dna strands)
C Elegans eats the e.coli and take up the dsRNA
The dsRNA of gene x is processed by Dicer in c elegans to make siRNA which will target and silence the gene x mRNA in the worm
Then see change in phenotype
For example the gene they knock down was the twitching gene in c elegans, this made them strat to twitch
What are small RNAs (sRNA) in prokaryotes
Regulate gene expression and plasmid replication, can turn on or off the activity of the mRNA
80-110 nt long
Encoded by small genes
Work by directly base paring with target mRNA to regulate their expression
What allows the binding to the sRNA to its target mRNA in prokaryotes
How do sRNA work
Hfq: RNA chaperone lets sRNA bind to target mRNA
Either do transactivatiom or repress:
Transactivation:
The mRNA transcript has the RBS (ribosome binding site) upstream of the coding region
The mRNA can fold into hairpin structure and block the RBS so ribosome can’t bind, but the sRNA binds to the loop and releases it from RBS
This turns on translation
Repression:
The sRNA bind to the RBS , blocks ribosome, repressor translation
How is crisper cas 9 similar to RNAi
They are both ways to regulate the expression of genes
What are riboswitches
Are part of the DNA and regulate transcriptional and translational gene expression upon changes in metabolites concentration
Have two components:
The expression platform: bound to the aptamer and contains the RBS
undergoes secondary structural changes upon aptamer binding to a metabolite
Aptamer: binds to small molecule metabolites and undergoes conformational change
This conformational change in the aptamer causes conformational changes in the expression platform
How it transcription termination regulated by the riboswitch
The aptamer has regions 1,2,3,4
In absence of the SAM metabolite regions 2 and 3 make a stem loop. The promoter region is open, the transcription is on
If SAM metabolite bound to aptamer, region 1 and 2 make stem loop, regions 3 and 4 make stem loop. Transcription is stopped
So binding of metabolite can regulate transcription of genes invoked in methionine or cysteine synthesis
How it translations termination regulated by the riboswitch
No metabolite, only region 2,3 stem loop, ribosome recognize RBS and translation occurs
Sam metabolite binding makes region 1,2 stem loop and 3,4 stem loop, RBS inaccessible, ribosome can’t bind, not translation
Riboswitch is
Regulatory RNA in prokaryotes and eukaryotes
