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Molecular Biology. > RNA Interference, Antisense, siRNA and miRNA. > Flashcards

RNA Interference, Antisense, siRNA and miRNA. Flashcards

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1
Q

Define the antisense strand?

A

A stand that is complementary to the coding strand.

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2
Q

Define gene knockout?

A

A process that helps to silence harmful genes that contribute to disease.

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3
Q

Define the guide strand?

A

The antisense strand on a pre-siRNA or a pre miRNA.

The guide strand will pair with the target mRNA.

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4
Q

Define the passenger strand?

A

The sense strand on a pre-sirRNA or a pre-miRNA.

This strand is discarded when the 2 strands separate.

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5
Q

What does RNAi mean?

A

An abbreviation of RNA interference.

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6
Q

Define a stem-loop?

A

The structure that is formed when single stranded RNA pairs with itself to create double stranded RNA.

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7
Q

Define transgenics?

A

The process of transferring genes into or out of a wild type species.

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8
Q

What does RNA interference refer to?

A

Ways that RNA synthesis can be modified.

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9
Q

What are the 3 types of RNA that are involved in RNA interference?

A

Antisense RNA.

siRNA (short interfering RNA).

miRNA (micro RNA).

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10
Q

How did the study of RNA interference arise?

A

In the quest to genetically engineer petunias to become a deeper shade of purple

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11
Q

What gene were scientists trying to overexpress when they were trying to genetically engineer petunias to become a deeper shade of purple?

A

The anthocyanin pigment gene.

This gene produces an enzyme called chalone synthase.

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12
Q

What happened when the anthocyanin pigment gene was overexpressed in purple petunias?

A

It formed a purple and white petunia or a petunia that was completely white.

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13
Q

Why was the overexpression of the anthocyanin pigment gene causing purple flowers to become white?

A

Because the wildtype contianed more APG mRNA than the modified plant.

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14
Q

What name did scientists give to the phenomenon that arose due to the overexpression of the anthocyanin pigment gene in purple petunias?

A

Co-supression as the amplification of APG suppressed both the endogenous gene and the transgenic gene.

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15
Q

What caused the supression of the anthocyanin pigment gene in the purple petunias?

A

RNA interference.

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16
Q

Which scientists discovered the phenomenon of RNA intereference?

A

Fire and Mello.

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17
Q

What experiment did Fire and Mello do to discover RNA interference?

A

They injected double stranded mRNA into of C.elegans gametes to try and generate mutant phenotypes.

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18
Q

What did Fire and Mello discover after they injected dsmRNA into C.elegans gametes?

A

That the endogenous DNA was degraded, meaning that its protein could not be produced.

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19
Q

How did Fire and Mello discover that the endogenous DNA was degraded in C.elegans gametes after injecting dsmRNA?

A

By staining mex-3 RNA so that its expression could be visualised.

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20
Q

What did Fire and Mello see when stained mex-r RNA and added the wildtype RNA in C.elegan gametes?

A

The dye highlighted that the gene had been normally expressed.

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21
Q

What did Fire and Mello see when stained mex-r RNA and added the antisense RNA in C.elegan gametes?

A

They found that the RNA was expressed at a reduced level.

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22
Q

What did Fire and Mello see when stained mex-r RNA and added the double stranded RNA in C.elegan gametes?

A

That no RNA was expressed at all and that the production of proteins in other cells were also affected.

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23
Q

Why did Fire and Mello describe the effects of adding different mRNA’s to C.elegans as RNA interference?

A

Because the addition of RNA into the system led to the changes.

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24
Q

Is it only genetic modifications that can induce genetic changes?

A

No.

The injections of various stands of RNA silence gene expression.

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25
Q

RNA is capable of regulating the expression of what?

A

The expression of genes.

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26
Q

Will the regulation of gene expression introduce positive or negative effects?

A

It can lead to the introduction of positive effects.

Or it can introduce negative effects that lead to disease.

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27
Q

What is an example of an RNA strand that is capable of regulating gene expression

A

A micro RNA (miRNA).

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28
Q

What are 3 common processes that can be regulated by micro RNA’s?

A

Metabolism.

Cell development.

Immune system activation.

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29
Q

How many human RNA’s are thought to be targets of RNA interference through the use of miRNA’s?

A

Around 1/3 .

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30
Q

What are the 3 classe’s of naturally occurring small RNA’s that can alter gene expression?

A

Micro RNA (miRNA).

Short interfering RNA (siRNA).

Repeat associated RNA (rasiRNA).

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31
Q

What are micro RNA’s derived from?

A

From specific double stranded pre-miRNA species that occur endogenously.

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32
Q

How can micro RNA’s regulate gene expression?

A

By repressing the translation of mRNA.

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33
Q

Where are short interfering RNA’S derived from?

A

From long dsRNAs that are exogenously injected into the cell e.g. by a virus.

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34
Q

How can short interfering RNA’s regulate gene expression?

A

By causing endogenous mRNA to be degraded.

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35
Q

How do repeat associated RNA’s differ from micro and siRNA’s?

A

They are longer than siRNA’s or miRNA’s.

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36
Q

How do repeat associated RNA’s regulate gene expression?

A

By causing the formation of heterochromatin which silence’s gene expression.

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37
Q

Is an siRNA endogenous or exogenous??

A

Exogenous.

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38
Q

What happens to an siRNA when it enters the cell?

A

The long strand of double stranded RNA is cut into short double stranded segments by an endoculease.

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39
Q

What are the small fragments known as after an sIRNA has been cleaved by an endonuclease?

A

As siRNA duplexes.

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40
Q

What happens to the siRNA duplexes once they have been formed?

A

They are separated into single strands by various enzymes.

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41
Q

What will the single stranded siRNA duplexes do once they have been formed in the cell?

A

They will bind to complimentary regions on an endogenous mRNA such as the 3-UTR.

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42
Q

How will siRNA affect translation the translation of the mRNA that have bound to?

A

It will repress the translation of the mRNA.

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43
Q

Where is the pre-cursor of an miRNA synthesised?

A

It is made endogenously, meaning that it is synthesised by the cell.

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44
Q

How is the pre-cursor of an miRNA synthesised?

A

As one long single strand.

Both strands are complimentary and a loop is formed at the bottom.

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45
Q

What happens if there are non-complimentary regions on a pre-cursor of miRNA?

A

They will form hairpin loops within the long strand.

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46
Q

What happens when the miRNA pre-cursor has been formed?

A

An endonuclease removes the loop at the bottom of the molecule and this forms an miRNA duplex.

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47
Q

Why is the loop removed from the bottom of the miRNA pre-cursor?

A

It separates the 2 strands.

48
Q

What happens when the 2 strands of a pre miRNA have been separated to form an miRNA duplex?

A

The single strands bind to the complimentary regions on an mRNA strand and inhibit translation.

49
Q

Is the complimentary binding between miRNA and the mRNA strand perfect?

A

No.

Small bubbles or loops appear on the miRNA strand where the sequence does not correctly match up.

50
Q

How do an miRNA and an siRNA differ in size?

A

They are both less than 20 nucleotides.

51
Q

How do an miRNA and an siRNA differ in the location that they are synthesised?

A

siRNA is syntheised exogenously.

miRNA is synthesised endogenously.

52
Q

How do an miRNA and an siRNA differ in the way that they bind to their mRNA?

A

siRNA forms an exact match.

miRNA does not form an exact match.

53
Q

How do an miRNA and an siRNA differ in the way that they repress the translation of the mRNA that they bind to?

A

siRNA cleaves the portion of mRNA that it binds to.

miRNA represses translation of the mRNA.

54
Q

Where can miRNA segments bind to on an mRNA molecule?

A

Anywhere.

55
Q

What percentage of miRNA’s are expressed independently (on their own)?

A

60%.

56
Q

What percentage of miRNA’s are expressed in clusters?

A

15%.

57
Q

What percentage of miRNA’s are expressed in introns?

A

25%.

58
Q

miRNA’s that are expressed in clusters are often found in what kind of cells?

A

In cancer cells.

59
Q

How are miRNA’s synthesised by the cell?

A

By an RNA polymerase II whcih will read particular genes in DNA.

60
Q

How are miRNA’s originally synthesised?

A

As long primary miRNAs (pri-miRNAs) which are complete with poly-A tails and 5 prime cap.

61
Q

What happens during miRNA synthesis after the pri-mRNA’s have been synthesised?

A

They are edited by the DROSHA protein which cleaves any unnecessary segments of miRNA to form pre-miRNA.

62
Q

What are notable features on a pre-miRNA?

A

A hairpin loop.

A 2 nucleotide sequence that forms a 3-prime overhang.

63
Q

What happens to the pre-miRNA once it has been formed during miRNA synthesis?

A

It is transported from the nucleus to the cytoplasm.

64
Q

What proteins transports the pre-miRNA from the nucleus to the cytoplasm during miRNA synthesis?

A

Exportin-5.

65
Q

What happens to the pre-miRNA when it is delivered to the cytoplasm during miRNA synthesis?

A

An endonuclease called DICER splits the dsRNA into single strands of around 20 nucleotides.

66
Q

What happens during miRNA synthesis after the short single strands have been formed by DICER?

A

DICER takes one of the miRNA strands and attaches it to the RNA silencing complex.

67
Q

What is the RNA silencing complex also known as?

A

The RISC factor.

68
Q

What will the RISC complex do once the miRNA strand is bought to it by DICER during miRNA synthesis?

A

It will imperfectly attach it the 3-UTR of the target mRNA.

69
Q

How does the attachment of miRNA to the 3-UTR of the target mRNA afect the translation of the mRNA?

A

It will stop the formation of the poly-A tails.

70
Q

How does the miRNA stopping the formation of the poly A tails on the target mRNA stop translation?

A

It prevents the formation of the lariat structure which stops the ribosome attaching to the mRNA.

71
Q

What kind of organism is used to make an siRNA?

A

An exogenous source such as a virus.

72
Q

How are pre-siRNA’s formed during siRNA sysnthesis?

A

The long strands of exogenous mRNA are cleaved into small double stranded fragments by the DICER protein.

73
Q

How are the 2 strands of pre-siRNA related to the target mRNA?

A

They are sense and antisense strands to the target mRNA.

74
Q

What is the antisense strand of a pre-siRNA known as?

A

As the guide strand.

75
Q

What is the sense strand of a pre-siRNA known as?

A

As the passenger strand.

76
Q

What strand of a pre-siRNA will bind to the mRNA of interest?

A

The guide strand.

77
Q

How does the guide strand of a pre-siRNA attach to the mRNA of interest?

A

The dicer protein presents it to the RISC complex which will then bind it to the target mRNA.

78
Q

Does the RISC protein contains endonuclease ability?

A

Yes.

79
Q

How does the siRNA silence the mRNA of interest?

A

The endonculease ability of the RISC protein cleaves the siRNA and the mRNA that it is bound to from the mRNA strand.

80
Q

What are the 5 functional domains within the DICER protein?

A

A dead box binding domain.

A PAZ domain.

An RNAase III domain.

An NLS domain.

A DRBD domain.

81
Q

What is the dead box binding domain of a DICER protein used for?

A

To unwind RNA.

82
Q

What energy does the dead box binding domain of a DICER protein use to unwind RNA?

A

ATP.

83
Q

What is the PAZ domain of a DICER protein used for?

A

For binding to an RNA strand.

84
Q

What is the RNAase III domain of a DICER protein used for?

A

It will lead to the formation of breaks in the double stranded RNA.

85
Q

What is the NLS domain of a DICER protein used for?

A

A nuclear localisation domain.

86
Q

What is the DRBD domain of a DICER protein used for?

A

It is the double stranded RNA interaction domain.

87
Q

What are the 3 known components of the RISC protein?

A

DICER protein.

Argonaute proteins.

Various RNA binding proteins.

88
Q

What argonaute proteins are found in humans?

A

They are called AGO-1 and AGO-2.

89
Q

What domains do the AGO-1 and AGO-2 argonaute proteins contain?

A

A PAZ domain which binds to small RNA sequences.

A PIWI domain which recognise’s double stranded RNA.

90
Q

What kind of RNA interference is AGO-1 used for?

A

For RNA interference that is directed by miRNA.

91
Q

What kind of RNA interference is AGO-2 used for?

A

For cleaving the target mRNA around 10 nucleotides from the binding site with an siRNA.

92
Q

What does the LIN-4 region in the C.elegans genome encode for?

A

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.

93
Q

What will RASI-RNA’s bind to to form a complex that induce’s the fomation of chromatin?

A

With chromatin remodelling enzymes such as DNA methyltransferase and histone de-acetylase.

94
Q

What is found in the complex that is responsible for inducing the formation of chromatin?

A

A RASI RNA.

The RNAi induced methylation complex (RIMC).

95
Q

How does RASI-RNA and RIMC complex lead to the formation of chromatin?

A

It leads the de-acetylation of histones and the methylation of cytosine residues which will form heterochromatin.

96
Q

How have siRNA’s helped to fight hepatitis B?

A

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.

97
Q

Nasal administration of siRNA’s helped to combat what virus in mice?

A

The influenza virus.

98
Q

How does the BCL-2 protein help a cell to resist apoptosis?

A

It inhibits a product called caspase which is responsible for inducing apoptosis?

99
Q

BCL-2 is often up-regulated in what kind of cells?

A

In cancer cells.

100
Q

How have scientists used RNAi to inhibit the production of the BCL-2 protein in cancer patients?

A

They use RNAi to cleave the mRNA that codes for BCL-2.

This means BCL-2 is not produced.

101
Q

How does the inhibition of BCL-2 help kill cancer cells?

A

It allows caspase levels to rise and apoptosis of the cancer cells to occur.

102
Q

The presence of miRNA’s will differ in which portions of the heart?

A

The miRNA’s in a diseased portion of the heart are different from the miRNAs in the healthy portion.

103
Q

How does heart disease effect the expression of miRNA?

A

It results in the different expressions of miRNA’s.

104
Q

How can the expression of different miRNA’s in the heart help to diagnose heart disease?

A

Samples can be taken from the heart and analysed for the presence of different miRNA’s.

105
Q

What 3 miRNA’s that are commonly found to be down regulated cancerous tissue?

A

miRNA-15a.

miRNA-16.

miRNA-21.

106
Q

What is the antisense strand of DNA or RNA?

A

The strand that is complimentary to the coding strand.

107
Q

What strand of DNA will the antisense strand of RNA bind to?

A

The sense strand.

108
Q

Which strand of DNA is responsible for coding for mRNA?

A

The sense strand.

109
Q

How can synthesised strands of DNA block gene expression?

A

They can bind to the coding strand of DNA and block the translation of mRNA.

110
Q

Synthetically synthesised antisense sequences of DNA are designed to do what?

A

To bid to the sense strand and inhibit the expression of a single gene.

111
Q

What diseases have synthetically synthesised antisense sequences of DNA been used to treat?

A

HIV.

Hepatitis B.

Various cancers.

112
Q

What are the differences in the types of RNA that are used in antisense RNA and RNAi?

A

Antisense RNA use’s single stranded RNA.

RNAi uses double stranded RNA.

113
Q

What are the differences in the use of cellular machinery during translational interference by antisense RNA and RNAi?

A

Antisense RNA does not use cellular machinery.

RNAi uses cellular machinery.

114
Q

What are the differences in efficiency of interference by antisense RNA and RNAi?

A

Antisense RNA is not very efficient.

The efficiency of RNAi depends on the availability of cellular machinery.

115
Q

What are the differences in the manufacture of the RNA in interference by antisense RNA and RNAi?

A

Antisense RNA is always made exogneously by scientists.

RNAi. Endogenous for miRNA. Exogenously made by scientists or viruses for siRNA.

Molecular Biology. (22 decks)

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