RNA Interference, Antisense, siRNA and miRNA. Flashcards
Define the antisense strand?
A stand that is complementary to the coding strand.
Define gene knockout?
A process that helps to silence harmful genes that contribute to disease.
Define the guide strand?
The antisense strand on a pre-siRNA or a pre miRNA.
The guide strand will pair with the target mRNA.
Define the passenger strand?
The sense strand on a pre-sirRNA or a pre-miRNA.
This strand is discarded when the 2 strands separate.
What does RNAi mean?
An abbreviation of RNA interference.
Define a stem-loop?
The structure that is formed when single stranded RNA pairs with itself to create double stranded RNA.
Define transgenics?
The process of transferring genes into or out of a wild type species.
What does RNA interference refer to?
Ways that RNA synthesis can be modified.
What are the 3 types of RNA that are involved in RNA interference?
Antisense RNA.
siRNA (short interfering RNA).
miRNA (micro RNA).
How did the study of RNA interference arise?
In the quest to genetically engineer petunias to become a deeper shade of purple
What gene were scientists trying to overexpress when they were trying to genetically engineer petunias to become a deeper shade of purple?
The anthocyanin pigment gene.
This gene produces an enzyme called chalone synthase.
What happened when the anthocyanin pigment gene was overexpressed in purple petunias?
It formed a purple and white petunia or a petunia that was completely white.
Why was the overexpression of the anthocyanin pigment gene causing purple flowers to become white?
Because the wildtype contianed more APG mRNA than the modified plant.
What name did scientists give to the phenomenon that arose due to the overexpression of the anthocyanin pigment gene in purple petunias?
Co-supression as the amplification of APG suppressed both the endogenous gene and the transgenic gene.
What caused the supression of the anthocyanin pigment gene in the purple petunias?
RNA interference.
Which scientists discovered the phenomenon of RNA intereference?
Fire and Mello.
What experiment did Fire and Mello do to discover RNA interference?
They injected double stranded mRNA into of C.elegans gametes to try and generate mutant phenotypes.
What did Fire and Mello discover after they injected dsmRNA into C.elegans gametes?
That the endogenous DNA was degraded, meaning that its protein could not be produced.
How did Fire and Mello discover that the endogenous DNA was degraded in C.elegans gametes after injecting dsmRNA?
By staining mex-3 RNA so that its expression could be visualised.
What did Fire and Mello see when stained mex-r RNA and added the wildtype RNA in C.elegan gametes?
The dye highlighted that the gene had been normally expressed.
What did Fire and Mello see when stained mex-r RNA and added the antisense RNA in C.elegan gametes?
They found that the RNA was expressed at a reduced level.
What did Fire and Mello see when stained mex-r RNA and added the double stranded RNA in C.elegan gametes?
That no RNA was expressed at all and that the production of proteins in other cells were also affected.
Why did Fire and Mello describe the effects of adding different mRNA’s to C.elegans as RNA interference?
Because the addition of RNA into the system led to the changes.
Is it only genetic modifications that can induce genetic changes?
No.
The injections of various stands of RNA silence gene expression.
RNA is capable of regulating the expression of what?
The expression of genes.
Will the regulation of gene expression introduce positive or negative effects?
It can lead to the introduction of positive effects.
Or it can introduce negative effects that lead to disease.
What is an example of an RNA strand that is capable of regulating gene expression
A micro RNA (miRNA).
What are 3 common processes that can be regulated by micro RNA’s?
Metabolism.
Cell development.
Immune system activation.
How many human RNA’s are thought to be targets of RNA interference through the use of miRNA’s?
Around 1/3 .
What are the 3 classe’s of naturally occurring small RNA’s that can alter gene expression?
Micro RNA (miRNA).
Short interfering RNA (siRNA).
Repeat associated RNA (rasiRNA).
What are micro RNA’s derived from?
From specific double stranded pre-miRNA species that occur endogenously.
How can micro RNA’s regulate gene expression?
By repressing the translation of mRNA.
Where are short interfering RNA’S derived from?
From long dsRNAs that are exogenously injected into the cell e.g. by a virus.
How can short interfering RNA’s regulate gene expression?
By causing endogenous mRNA to be degraded.
How do repeat associated RNA’s differ from micro and siRNA’s?
They are longer than siRNA’s or miRNA’s.
How do repeat associated RNA’s regulate gene expression?
By causing the formation of heterochromatin which silence’s gene expression.
Is an siRNA endogenous or exogenous??
Exogenous.
What happens to an siRNA when it enters the cell?
The long strand of double stranded RNA is cut into short double stranded segments by an endoculease.
What are the small fragments known as after an sIRNA has been cleaved by an endonuclease?
As siRNA duplexes.
What happens to the siRNA duplexes once they have been formed?
They are separated into single strands by various enzymes.
What will the single stranded siRNA duplexes do once they have been formed in the cell?
They will bind to complimentary regions on an endogenous mRNA such as the 3-UTR.
How will siRNA affect translation the translation of the mRNA that have bound to?
It will repress the translation of the mRNA.
Where is the pre-cursor of an miRNA synthesised?
It is made endogenously, meaning that it is synthesised by the cell.
How is the pre-cursor of an miRNA synthesised?
As one long single strand.
Both strands are complimentary and a loop is formed at the bottom.
What happens if there are non-complimentary regions on a pre-cursor of miRNA?
They will form hairpin loops within the long strand.
What happens when the miRNA pre-cursor has been formed?
An endonuclease removes the loop at the bottom of the molecule and this forms an miRNA duplex.
Why is the loop removed from the bottom of the miRNA pre-cursor?
It separates the 2 strands.
What happens when the 2 strands of a pre miRNA have been separated to form an miRNA duplex?
The single strands bind to the complimentary regions on an mRNA strand and inhibit translation.
Is the complimentary binding between miRNA and the mRNA strand perfect?
No.
Small bubbles or loops appear on the miRNA strand where the sequence does not correctly match up.
How do an miRNA and an siRNA differ in size?
They are both less than 20 nucleotides.
How do an miRNA and an siRNA differ in the location that they are synthesised?
siRNA is syntheised exogenously.
miRNA is synthesised endogenously.
How do an miRNA and an siRNA differ in the way that they bind to their mRNA?
siRNA forms an exact match.
miRNA does not form an exact match.
How do an miRNA and an siRNA differ in the way that they repress the translation of the mRNA that they bind to?
siRNA cleaves the portion of mRNA that it binds to.
miRNA represses translation of the mRNA.
Where can miRNA segments bind to on an mRNA molecule?
Anywhere.
What percentage of miRNA’s are expressed independently (on their own)?
60%.
What percentage of miRNA’s are expressed in clusters?
15%.
What percentage of miRNA’s are expressed in introns?
25%.
miRNA’s that are expressed in clusters are often found in what kind of cells?
In cancer cells.
How are miRNA’s synthesised by the cell?
By an RNA polymerase II whcih will read particular genes in DNA.
How are miRNA’s originally synthesised?
As long primary miRNAs (pri-miRNAs) which are complete with poly-A tails and 5 prime cap.
What happens during miRNA synthesis after the pri-mRNA’s have been synthesised?
They are edited by the DROSHA protein which cleaves any unnecessary segments of miRNA to form pre-miRNA.
What are notable features on a pre-miRNA?
A hairpin loop.
A 2 nucleotide sequence that forms a 3-prime overhang.
What happens to the pre-miRNA once it has been formed during miRNA synthesis?
It is transported from the nucleus to the cytoplasm.
What proteins transports the pre-miRNA from the nucleus to the cytoplasm during miRNA synthesis?
Exportin-5.
What happens to the pre-miRNA when it is delivered to the cytoplasm during miRNA synthesis?
An endonuclease called DICER splits the dsRNA into single strands of around 20 nucleotides.
What happens during miRNA synthesis after the short single strands have been formed by DICER?
DICER takes one of the miRNA strands and attaches it to the RNA silencing complex.
What is the RNA silencing complex also known as?
The RISC factor.
What will the RISC complex do once the miRNA strand is bought to it by DICER during miRNA synthesis?
It will imperfectly attach it the 3-UTR of the target mRNA.
How does the attachment of miRNA to the 3-UTR of the target mRNA afect the translation of the mRNA?
It will stop the formation of the poly-A tails.
How does the miRNA stopping the formation of the poly A tails on the target mRNA stop translation?
It prevents the formation of the lariat structure which stops the ribosome attaching to the mRNA.
What kind of organism is used to make an siRNA?
An exogenous source such as a virus.
How are pre-siRNA’s formed during siRNA sysnthesis?
The long strands of exogenous mRNA are cleaved into small double stranded fragments by the DICER protein.
How are the 2 strands of pre-siRNA related to the target mRNA?
They are sense and antisense strands to the target mRNA.
What is the antisense strand of a pre-siRNA known as?
As the guide strand.
What is the sense strand of a pre-siRNA known as?
As the passenger strand.
What strand of a pre-siRNA will bind to the mRNA of interest?
The guide strand.
How does the guide strand of a pre-siRNA attach to the mRNA of interest?
The dicer protein presents it to the RISC complex which will then bind it to the target mRNA.
Does the RISC protein contains endonuclease ability?
Yes.
How does the siRNA silence the mRNA of interest?
The endonculease ability of the RISC protein cleaves the siRNA and the mRNA that it is bound to from the mRNA strand.
What are the 5 functional domains within the DICER protein?
A dead box binding domain.
A PAZ domain.
An RNAase III domain.
An NLS domain.
A DRBD domain.
What is the dead box binding domain of a DICER protein used for?
To unwind RNA.
What energy does the dead box binding domain of a DICER protein use to unwind RNA?
ATP.
What is the PAZ domain of a DICER protein used for?
For binding to an RNA strand.
What is the RNAase III domain of a DICER protein used for?
It will lead to the formation of breaks in the double stranded RNA.
What is the NLS domain of a DICER protein used for?
A nuclear localisation domain.
What is the DRBD domain of a DICER protein used for?
It is the double stranded RNA interaction domain.
What are the 3 known components of the RISC protein?
DICER protein.
Argonaute proteins.
Various RNA binding proteins.
What argonaute proteins are found in humans?
They are called AGO-1 and AGO-2.
What domains do the AGO-1 and AGO-2 argonaute proteins contain?
A PAZ domain which binds to small RNA sequences.
A PIWI domain which recognise’s double stranded RNA.
What kind of RNA interference is AGO-1 used for?
For RNA interference that is directed by miRNA.
What kind of RNA interference is AGO-2 used for?
For cleaving the target mRNA around 10 nucleotides from the binding site with an siRNA.
What does the LIN-4 region in the C.elegans genome encode for?
A 22 nucleotide non-coding RNA strand that bind to the RISC factor.
This complex can bind to 7 different sites on the 3-UTR of the LIN-14 gene which prevents ribosomal assembly.
What will RASI-RNA’s bind to to form a complex that induce’s the fomation of chromatin?
With chromatin remodelling enzymes such as DNA methyltransferase and histone de-acetylase.
What is found in the complex that is responsible for inducing the formation of chromatin?
A RASI RNA.
The RNAi induced methylation complex (RIMC).
How does RASI-RNA and RIMC complex lead to the formation of chromatin?
It leads the de-acetylation of histones and the methylation of cytosine residues which will form heterochromatin.
How have siRNA’s helped to fight hepatitis B?
The use siRNA’s to fight hepatitis B resulted in fewer viral copies. The injection of the apo-lipoprotien-B siRNA into mice helped to reduce cholesterol and resulted in gene knockout. The electropulsation of siRNA’s into mice also resulted in gene knockout.
Nasal administration of siRNA’s helped to combat what virus in mice?
The influenza virus.
How does the BCL-2 protein help a cell to resist apoptosis?
It inhibits a product called caspase which is responsible for inducing apoptosis?
BCL-2 is often up-regulated in what kind of cells?
In cancer cells.
How have scientists used RNAi to inhibit the production of the BCL-2 protein in cancer patients?
They use RNAi to cleave the mRNA that codes for BCL-2.
This means BCL-2 is not produced.
How does the inhibition of BCL-2 help kill cancer cells?
It allows caspase levels to rise and apoptosis of the cancer cells to occur.
The presence of miRNA’s will differ in which portions of the heart?
The miRNA’s in a diseased portion of the heart are different from the miRNAs in the healthy portion.
How does heart disease effect the expression of miRNA?
It results in the different expressions of miRNA’s.
How can the expression of different miRNA’s in the heart help to diagnose heart disease?
Samples can be taken from the heart and analysed for the presence of different miRNA’s.
What 3 miRNA’s that are commonly found to be down regulated cancerous tissue?
miRNA-15a.
miRNA-16.
miRNA-21.
What is the antisense strand of DNA or RNA?
The strand that is complimentary to the coding strand.
What strand of DNA will the antisense strand of RNA bind to?
The sense strand.
Which strand of DNA is responsible for coding for mRNA?
The sense strand.
How can synthesised strands of DNA block gene expression?
They can bind to the coding strand of DNA and block the translation of mRNA.
Synthetically synthesised antisense sequences of DNA are designed to do what?
To bid to the sense strand and inhibit the expression of a single gene.
What diseases have synthetically synthesised antisense sequences of DNA been used to treat?
HIV.
Hepatitis B.
Various cancers.
What are the differences in the types of RNA that are used in antisense RNA and RNAi?
Antisense RNA use’s single stranded RNA.
RNAi uses double stranded RNA.
What are the differences in the use of cellular machinery during translational interference by antisense RNA and RNAi?
Antisense RNA does not use cellular machinery.
RNAi uses cellular machinery.
What are the differences in efficiency of interference by antisense RNA and RNAi?
Antisense RNA is not very efficient.
The efficiency of RNAi depends on the availability of cellular machinery.
What are the differences in the manufacture of the RNA in interference by antisense RNA and RNAi?
Antisense RNA is always made exogneously by scientists.
RNAi. Endogenous for miRNA. Exogenously made by scientists or viruses for siRNA.
