Topic 12 Regulatory RNAs

Question 1

What are regulatory rna

What is RNAi

Answer 1

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

Question 2

What are the categories of RNAi machinery

When we’re they discovered

Answer 2

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

Question 3

What are the key components that make RNAi machinery (non coding rna)

Answer 3

Dicer

Microprocessor complex

RNA induced silencing complex

Argonaute protien

Question 4

What is dicer

Answer 4

An RNase-III like enzyme that processes long dsRNA to make siRNA or stem loop structures to make miRNA

Question 5

What is the microprocessor complex

Answer 5

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

Question 6

Whag is the formation of non coding RNA like miRNA and siRNA called

Answer 6

Biogenesis

Question 7

What is RISC

What is the argonaute protien

Answer 7

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)

Question 8

Slode 5 note

Answer 8

Idk

Question 9

Decribe how siRNA undergoes biogenesis

Answer 9

1.long dsRNA introduced to the cells through foreign particles (viruses)

2. Dicer chops the long dsRNA to short dsRNA (22nt), this dsRNA is loaded into the RISC complex
3. When on RISC complex, the two strands of dsRNA melt and becomes two separate strands
4. 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

5. the siRNA guide strand compliments the sequence of the target mRNA which activates the Argonaute to degrade the target mRNA
6. No gene expression since mRNA degraded

Question 10

Decribe how miRNA undergoes biogenesis

Answer 10

MicroRNA need to undergo multiple cleavages

1. 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

2. 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
3. The 22 nt miRNA binds to miRNA risc in the cytoplasm
4. The stem loop structure disscosistes and becomes passenger strand and guide strand
5. The miRNA is not 100% compliment to the targeting RNA

Question 11

Whag is the functional difference between miRNA and siRNA

Answer 11

SiRNA is 100% complaints to its target RNA, miRNA isn’t

SiRNA and miRNA have their own separate risc complexes that they bind to

Question 12

How does the RNAi silencing guide rna promote gene expression silencing

Answer 12

1. MRNA degradation
2. Translational repression
3. Return to nucleus and silence genes by directing chromatin modifications to make heterochromatin (transcription is repressed)

Question 13

Describe the structure of miRNA

Answer 13

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

Question 14

How can miRNA be found by bioinformatics

Answer 14

Can look at rna sequence and see if they have seuwences that fold back to form a stem loop structure

Question 15

What sequences encode pre-miRNA

Answer 15

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

Question 16

What are seed residues

Answer 16

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

Question 17

What techniques can be used to detect prescience of non coding regulatory rna like siRNA and miRNA

Answer 17

Northern blot and qPCR

Question 18

Describe the structure of pri-miRNA

How many turn of helix is mature miRNA

Answer 18

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

Question 19

Describe how the microprocessor complex cleaves pri-MIRNA to pre miRNA

Answer 19

Happens in nucleus

1. Pasha (recognition subunit) and drosha (catalytic subunit) amale ip the microprocessor complex and recognize the pri-miRNA
2. Drosha makes two cleavages at the ss and ds rna junction
3. 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

4. The pre-miRNA 3’ overhang is important for Dicer recognition in the cytoplasm

Question 20

Describe the structure of Dicer

Answer 20

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

Question 21

What is similar in Drosha and Dicer

Answer 21

They are both RNase III enzymes that cleave miRNA

Question 22

How does the Dicer cleave the pre miRNA

Answer 22

The premirna is sent to cytoplasm

The cleavage occurs and the pre miRNA becomes 22 nt long mature mi rna

Question 23

How does the guide miRNA get formed and risc get activated in miRNA biogenesis

Answer 23

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

2. DsRNA goes to risc (now called miRISC)

dsRNA is denatured into a single guide strand and passenger strand

3. Passenger strand is degraded, guide strand recognizes the target RNA and guides risc to it
4. RISC with the single guide miRNA is the mature risc
5. The guide RNA recognizes and base pairs with target mRNA

Question 24

What is the structure of the Argonaute

Answer 24

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

Question 25

Explain how the RISC complex cleaves the target mRNA using miRNA

Answer 25

1. The guide rna brings the miRISC to the target mRNA 2. Guide stand non perfectly compliments the 3’ UTR mRNA sequence because of the seed sequence so we see bulges 3. This binding of the guide strand sends signal to the Argonaute to degrade the target mRNA through its RNase H activity 4. Since rna is chopped, gene silence no protien made, low gene expression

Question 26

Explain how the RISC complex cleaves the target mRNA using siRNA

Answer 26

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

Question 27

How does the guide strand bind to the RISC compelx What types of cleavage does the microprocessor complex do

Answer 27

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

Question 28

Overall in function how is siRNA diff from mi rna

Answer 28

SiRNA is more precise for the mRNA target, miRNA has more ambiguity because less complimentarity so it leads to off target effects

Question 29

What other ways can risc silence genes

Answer 29

Inhibits translation 1. by the miRNA sequestering the target mRNA to processing bodies. These processing bodies (P-bodies/p-granules) are in the cytoplasm and repress translation 2. 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 3. Introduce chromatin modifications to make heterchromatin, transcription gene silencing because packed too tightly

Question 30

Explain how gene silencing by chromatin modification can happen using regulatory rna

Answer 30

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

Question 31

What are the biological functions of RNAi

Answer 31

1. Helps against viral infection in plants and humans 2. 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

Question 32

Describe the first phenomena of RNAi

Answer 32

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

Question 33

How can RNAi be used as a research tool

Answer 33

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

Question 34

What are small RNAs (sRNA) in prokaryotes

Answer 34

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

Question 35

What allows the binding to the sRNA to its target mRNA in prokaryotes How do sRNA work

Answer 35

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

Question 36

How is crisper cas 9 similar to RNAi

Answer 36

They are both ways to regulate the expression of genes

Question 37

What are riboswitches

Answer 37

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

Question 38

How it transcription termination regulated by the riboswitch

Answer 38

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

Question 39

How it translations termination regulated by the riboswitch

Answer 39

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

Question 40

Riboswitch is

Answer 40

Regulatory RNA in prokaryotes and eukaryotes