GILESTRO - rnai

Question 1

Initial intuition:

Answer 1

RNA transcription and translation are steric activities - arise from the spatial arrangement of 3D structures. RNA itself is also a steric molecule – not a flat coding sequence; it forms complex 2D secondary structures to regulate translation & bind with proteins.

Idea: to use antisense RNA that bind with any region of the ss mRNA, making a ds molecule which will interfere with translation by disrupting the spatial arrangement of the structures involved.

Question 2

Early experiments with RNAi

Answer 2

Experiment with Thymidine Kinase gene:
* Insertion of antisense RNA actually does reduce the expression of target protein but are needed in high conc and occur at low efficiency (never 0% suppression)

DNA Plant technology corp. experiment with chalcone synthase – gene in petunia flower important for its purple color
* results in unpredictable products with various different petal colors & patterns

Experiment with UNC gene – involved with the function of C. elegans nervous system
* Results in disrupted movement
* BUT apart from antisense, the sense RNA added also works – the reason is unclear which goes against the hypothesis of disruption caused by antisense binding to mRNA

Experiment with Par-1 gene in C. elegans related to asymmetric cell division which leads to cell polarity
* Results in gene disruption upon insertion of BOTH sense & antisense (even higher when sense is used)

Question 3

Uncovering the reason behind gene disruption caused by sense RNA

Answer 3

Observation:
* Get phenotype only when inject BOTH sense and antisense RNA
o The mixture of sense + antisense was at least 100 times stronger than the single
sense or antisense
* Injecting the two strands with an interval in between led to no silencing
o need to inject complementary sense and antisense together to form dsRNA
* Co-injection of dsRNA unrelated to unc-22 did not potentiate the effect of single unc-22 strands
o Need to use the dsRNA that is complementary to the target gene
o Does not work if use 1 antisense complementary to target gene & also use
dsRNA that target another region – the unrelated dsRNA is not involved in
activating another machinery – shows sequence-specificity
* dsRNA targeting promoter or introns did not lead to silencing
o suggests that the process happens outside of the nucleus – process exerts an
effect on processed mRNA outside nucleus that has already matured and spliced (lose intron and promotor activity)
* The lowest dose of dsRNA tested (~60k molecules per adult) still led to phenotype in 100 progenies.
o certainly not enough molecules but can still pass down!
o dsRNA detected and purified from the progeny was shown to retain silencing
activity – reinjection of dsRNA isolated from progeny still leads to silencing
* mRNA of targeted genes was greatly reduced in expression – not just protein level
o suggests that the inserted dsDNA is not just interfering with the translation process – also directly interferes with mRNA

Leads to new intuition of RNAi
* Since dsRNA does not exist in natural cells, when they are injected into the cell, the cell responds by destroying the actual acting ss mRNA

Question 4

Identifying molecules involved in the new RNAi intuition

By 2 approaches:
* Genetic
* Biochemical (mostly discovered by biochemical approach)

Answer 4

Genetic approach – mutate worm to express GFP
1. perform RNAi on GFP
2. affected worm will lose GF
3. perform random mutagenesis on worms that have lost GF to see which affected worm will regain GF – regain of GF indicates that the mutation disrupts with the machinery involved in RNAi
4. identify the disrupted - is the gene involved with RNAi machinery

Biochemical approach
1. Inject RNAi into the cell (target any gene)
2. Crush cells to extract protein
3. Performing pull-down experiment using beads with affinity to the RNAi to observe which protein is pulled down with it
* discovered dicer

Question 5

The RNAi pathway:

Answer 5

1. introduced dsRNA is cleaved by dicer
2. helicase unwinds dsRNA fragments
3. one of the strands is selected to load onto Argonaut protein which associates with other proteins to form RISC
   (RNA-inducing silencing complelx)
4. the selected ssRNA will then direct RISC to the target complementary mRNA & induce argonaut-directed cleavage of the mRNA

Question 6

2 types of dsRNA used in RNAi

Answer 6

1. small interfering RNA (siRNA)
   * usually exogenous dsRNA from viruses or derived experimentally in labs
2. microRNA (miRNA)
   * derived from RNA transcribed in the nucleus that have ds region due hairpin structure formation

Question 7

Detailed RNAi steps:

Answer 7

1. Biogenesis of miRNA & siRNA from pre-cursors
2. The miRNA/ siRNA fragment are loaded onto Argonaut
3. Strand selection:
   * 1 strand is selected to remain bound to argonaut (guide strand) & the other strand (passenger strand) is degraded
4. miRNA/siRNA-bound Argonaut associates with other proteins to form the RISC
5. Due to complementarity between the miRNA/siRNA and the target sequence, RISC is directed to the target sequence
6. Effects on the target sequence depend on the level of complementarity of the dsRNA with the target sequence
   * 100% complementarity will lead to Argonaut-catalyzed mRNA degradation
   * Partial complementarity (imprecise matching) will lead to translation inhibition
   * siRNA often have perfect complementary to target sequence while only a part of miRNA known as SEED will pair with the target mRNA
7. After cleavage/ translation inhibition is done, miRNA/ siRNA can be recycled by returning to form another RISC complex

Question 8

Biogenesis of miRNA & siRNA from pre-cursors

Answer 8

miRNA biogenesis:
1 A region of the transcribed genomic DNA produces dsRNA due to hairpin structure formation
2 In the nucleus, Drosha cleaves out the other ss regions of the RNA to form the smaller pre-miRNA which is exported out of the nucleus by exportin 5
3 In the cytoplasm, Dicer cleaves the loop region connecting the complementary sequences of the pre-miRNA to form mature miRNA duplex

siRNA biogenesis:
Dicer perform 2 cuts on the long ds siRNA into smaller fragments (21-22 nt
long)
* In lab experiments with human cells, injected siRNA are often already 21-22 nt long to bypass the dicer step because it is more difficult to introduce dsRNA into the cell membrane – if long = might not enter human cells

SO differences: miRNA = initial cut by drosha then 2nd cut by dicer, siRNA = both cuts by dicer

Question 9

RNAi as a defense mechanism for protection against RNA viruses

Answer 9

1 Virus dsRNA invades and try to hijack cell to produce its viral proteins & to replicate to create more viral particles
2 Dicer can be activated as an immune system response to cleave ds viral RNA BUT that cleavage also forms siRNA from viral dsRNA & is then loaded on to RISC
3 The siRNA generated will have full complementarity with other viral dsRNA so it will guide RISC & lead to more viral dsRNA degradation

Question 10

Mechanism of translation inhibition:

Answer 10

1. RISC lead to ribosome drop-off
   * ribosome dissociate from mRNA and stalls translation
2. Block circularization of mRNA
   * translation is done on circularized mRNA – efficient for ribosome to go from one end to the other continuously
   * no circularization slows down translation
3. Interfere with ribosome complex formation by competing with the 40S subunit for the 60s/elF6 complex.
   * eLF6 is a translation initiation factor that binds to the 60S ribosomal subunit. eLF6 is required to dissociate for the 60S association with 40S ribosomal subunit to form the 80S initiation complex. RISC prevents eIF6 dissociation thus the 80S initiation complex can’t be formed
4. Compete for translation initiation factors (eLF4F) binding to 5’ cap

Question 11

Translation inhibition instead of complete mRNA degradation allows for:

Answer 11

• Temporary silencing of a gene at a precise time during development, so mRNA is not completely destroyed and is available for protein production to resume afterwards
  o Thus, can study genes crucial for development of animal that are not able to
  completely remove from the early stage of development
• Prevent waste of energy if cells just produce mRNA that are all degraded
  SO, RNAi can target hundreds of endogenous mRNA (including targeting multiple genes in the same family & targeting of multiple areas of the same gene)

Question 12

Other types of small RNAs that can perform RNAi:

Answer 12

Ex: Secondary siRNA & tasiRNA
* dsRNA found only in c. elegans – allows signal amplification
* has RNA dependent RNA pol (RdRP)
o the ss siRNA binding to target mRNA can also act as a primer for the RdRP
o RdRP can elongate the dsRNA which can be further cleaved by Dicer
o Get more siRNA from more region of 100% complementarity apart from the initial siRNA
o Explains the observed effect of interference in 100s of C. elegans progeny even though only a small dose of RNAi was used

Question 13

RNAi screening (reverse genetics) – of a specific target gene/ large-scale RNAi screening

Methods of RNAi distribution into different systems

Answer 13

In humans & drosophila, have multi-well plate of cells
* the dsRNA is distributed into each well
(humans = 21-23 nt long siRNA, drosophila = long dsRNA which is later acted on by dicer)

In c. elegans:
* transform e. coli with plasmids containing target sequence flanked with T7 promotors on both strands
* E. coli will then produce both sense & antisense RNA which will be complementary, thus forming a dsRNA of the target sequence
* Infect C. elegans by putting the transformed e. coli on to its feeding plate

• UAS/GAL4 system can also be utilized for drosophila
  o sequence downstream of UAS designed to inverted repeats of the target gene so its RNA will form a hairpin structure. miRNA of target sequence is then formed after being acted on by Drosha & Dicer.

Question 14

Evidence of RNAi by GAL4/UAS system in Drosophila

Answer 14

• RNAi by GAL4/UAS system results in the same phenotype as mutants = successful effect

Modified transformer 2 (tra2) gene in RNAi and mutant females anatomically resemble males, including male genitalia and abdominal pigmentation

Modified eyes absent (eya) gene in RNAi and mutant males result in greatly reduced or absent eyes

Modified stubble (Sb) gene in RNAi and mutant males have short, stubby bristles on the notum

RNAi using induced production of miRNA by gal4/UAS system = cause knockdown: reduce expression by 90% – not 100% gene knockout
* Can use GAL4 system that is on and off ex. GAL4 expression which varies with temp (ex. if use GAL4 that is only active at 25°C but inactive at 18°C, can move flies from 18°C to 25°C only when we want to activate the interference

Question 15

Phenotypes screened using RNAi

Answer 15

• Muscle formation and development (flying muscles)
• Pain (from larvae to mice)
• Heart development and failure
• Bristle formation (asymmetric cell division)
• Obesity
• Immune response
• Sugar vs protein food preference
• Neuronal control of Drosophila courtship

Question 16

Bristle formation

Answer 16

• Bristle = important sensory organ for animals – used to detect sensory information (ex. gustatory input, airflow as they fly – indicate if flying in the right direction)
• Form as a hair which is held in place by a socket & has a neuron that enclosed by a sheath touching the hair
  o All components are derived from same precursor (SOP cell)
  o SOP undergoes asymmetric cell division – where mitosis of SOP cell gives rise to daughter cells that are different from mother cell – results in cell differentiation
  § Asymmetric cell division of SOP forms P2A & P2B
  § P2A undergoes asymmetric cell division again into hair and socket
  § P2B undergoes asymmetric cell division again into glia & a precursor cell
  § glia dies off while the precursor gives rise to the neuron and sheath
• Can use large scale RNAi screening to identify all the genes involved with this asymmetric cell division process
  o Introduce RNAi of many different genes using Genome-wide siRNA library
  o select for mutants with modified bristle, whose genes that involved with asymmetric cell division will be modified
  o Identification of the modified genes will provide info about the genes involved in the process

Question 17

Sugar vs protein food preference

Answer 17

1. Give flies a choice between purple food (protein rich) & blue food (sugar rich)
2. Check which food flies prefer
3. Perform large scale RNAi screening on the flies and repeat experiment to
   observe any change in food preference
   * Gives input on which gene regulates nutrition in the organism, what feedback loops the organisms use to regulate starvation, provide info for whether preference of food is dependent on sex
   o Females need to produce embryos which is an energetically costly process so
   females need greater caloric food, resulting in females preference of more
   protein rich food than males
   o Similar to why female mosquitos feed on human blood - because human blood
   contains protein – understanding the genes that regulate female mosquito’s
   preference of blood can be a good mechanism to stop malaria à develop a drug that targets that gene

Question 18

Natural RNAi involved in epigenetics:

Answer 18

X inactivation – females inactivate 1 copy of their X chromosome to generate = appropriate level of gene expression
* involves Xist - lncRNA that acts in cis to bind & recruit PRC2 to causes H3K27 trimethylation on the X chromosome it is transcribed from, which will repress the X
chromosome (turn it into closed chromatin)
* Nearby, also have Tisx, Xist antisense partner
* Due to complementarity between Xist & Tsix RNA, a dsRNA can be formed which is then acted on by dicer, thus fragmenting the RNA & resulting in Xist repression
* Thus, presence of Tisx inhibits Xist-mediated X inactivation via miRNA processing pathway