Antisense RNA L20-21

Question 1

What is antisense?

Answer 1

Antisense are generally shorter fragments of nucleic acid that are complimentary to the sense strand of the mRNA

Question 2

Sense mRNA codes for protein translation.

Therefore, antisense strand = ______ strand

Answer 2

Template

Question 3

We can use antisense mRNA to bind to sense mRNA to regulate its ____1____ and ____2____. Turn on and off.

Answer 3

1. Transcription
2. Translation

Question 4

Regulation of eukaryotic gene expression

1, 2, 3 & 4 = points of ______

Answer 4

Regulation

Question 5

Describe capping.

What does capping do?

Answer 5

Capping = adding a 5’ methyl-guanosine to the mRNA

• Helps to stop mRNA from being degraded by ribonucleases
• Acts as a site for initiation of translation

Question 6

What is splicing?

Answer 6

The removal of introns to produce the mature RNA to be translated.

Question 7

Describe polyadenylation.

What does polyadenylation do?

Answer 7

Polyadenylation = leads to cleaving of the protein at the 3’ site and adding of a polyadenosine tail.

It stabilises the mRNA.

Question 8

What is gene editing?

Answer 8

Gene editing = single amino acid insertions, deletions, mutations etc.

Question 9

Describe nuclear export.

Answer 9

Nuclear export = The transport of the mature mRNA from the nucleus to ribosomes.

Question 10

Introns and exons can be any length, therefore we need a mechanism to enable our splicing machinery to know where to cut.

This mechanism are the 5’ and 3’ ______ ______ ______.

Answer 10

Conserved splice sequences

Question 11

Describe the two-step splicing of introns.

Answer 11

Step 1

• Cut at 5’ splice site
• Creation of bond between 5’ end of intron and branch site

Step 2

• Cut at 3’ splice site to release intron lariat
• Ligation of two exons

Question 12

Naturally, antisense prevents the ____1____ machinery from binding to the mRNA thus blocking (turning off) the ____2____.

Answer 12

1. Translation
2. Gene

Question 13

Describe Thalassaemia.

What causes it?

Answer 13

Called Mediterranean anaemia. Essentially a blood disorder where the haemoglobin is not processed properly. Leads to all sorts of anaemia like symptoms.

Caused by cryptic splicing.

Question 14

What is a cryptic splice site?

How can we treat this?

Answer 14

Usually unused splice sites but if we get mutations in our gene, these cryptic splice sites can be activated. These leads to the translated protein being prematurely spliced, leading to a truncated protein being produced.

To prevent this we can use antisense RNA that will bind to the cryptic splice site mRNA thereby blocking it and prevent its translation.

Question 15

Duchenne Muscular Dystrophy

A C→T mutation causes a stop codon to be present in the middle of the gene. This leads to a ____1____ protein (dystrophin) being produced in sufferers. To eliminate the symptoms of Duchenne Muscular Dystrophy we can use ____2____ RNA to block this UGA (stop) codon and the exon it resides in thereby allowing a shortened dystrophin to be produced. It may be shortened but it still has greater functionality that the ____1____ form as exon 23 was found to not be vital for the proteins activity.

Answer 15

1. Truncated
2. Antisense

Question 16

Spinal Muscular Atrophy is a wasting disease where the neurons don’t form properly. Often fatal to infants.

Sometimes for splicing we need enhancers.

In exon 7 of the SMN protein there is an exonic enhancer of splicing.

SF2/ASF = Splicing Factor 2 / Alternative Splicing Factor

In normal SMN, SF2 binds to the splice site and initiates translation.

In the disease, there is a mutation in the enhancer leading to the splice site being skipped. This leads to exon 7 not being translated. A truncated -6,8- protein is produced = unstable SMN = disease phenotype.

So how can we treat this?

Answer 16

With antisense, we can remove the splice site protein and attach it to a piece of antisense RNA which exactly matches the mutated enhancer site. The protein is then put back into the system where it integrates back to where it originally was.

Splicing can then occur as normal and the normal SMN protein is produced = no disease phenotype.

Question 17

List the problems with antisense. (3)

Answer 17

• RNAs are relatively unstable (attacked by ribonucleases etc.)
• Difficult to transfer into cell
• Natural mechanisms in the cell block antisense function or degrade

Question 18

Normal DNA = Base (B), Deoxyribose sugar and phosphate backbone. Can be degraded – is usually degraded at the phosphate-sugar bond.

But we can fool the cell machinery into not degrading it by producing synthetic oligonucleotides by replacing a part of the ____1____ or ____2____ groups. This stops the cell from recognising it as DNA, thereby preventing its degradation.

RNA, that is more unstable due to having a 2’ OH group instead of a 2’ hydrogen on the sugar group. This enables self-cleavage and therefore degradation. So what we can do with synthetic RNAs is to change the 2’ OH group, for e.g. normally to a ____3____ or ____4____ group.

Answer 18

1. Phosphate
2. Sugar
3. Methyl
4. Ethyl

Question 19

What are the benefits of modified nucleic acids? (3)

Answer 19

Stability

• Modifying the sugar phosphate backbone reduces the rate of turnover

Delivery

• Modification results in molecules that are easier for cellular uptake

Not recognised as nucleic acids

• Important as the cellular machinery doesn’t sequester the antisense

Question 20

What is a ribozyme?

Answer 20

Catalytic RNA

Question 21

The hammerhead ribozyme

• Naturally occurring ribozyme that is present in several ____1____ RNA viruses
• Used to cleave individual genomic RNAs during rolling circle ____2____
• Naturally a cis-cleavage but can cleave in trans

Answer 21

1. Plant
2. Replication

Question 22

We can make ribozymes that don’t have to self-cleave. We can produce ____1____-ribozymes that can cleave things other than themselves. We can do this by designing the RNA ribozyme to bind to the specific target site that we wish to cleave (e.g. pathogenic RNA virus). The ribozyme binds through the ____2____ of antisense. When bound, the ribozyme forms its shape, then the active site is able to interact and cleave the target sequence.

Answer 22

1. Trans
2. Complementarity

Question 23

An aptamer is effectively an artificial ______.

Answer 23

Antibody

Question 24

When using SELEX to synthesize DNA aptamers, how can we separate the dsDNA to ssDNA?

Why does it need to be separated?

Answer 24

To ensure this, one of the primers contains a biotin molecule at its extremity. We can then pull the biotin down on streptavidin beads to separate out the ssDNA with the biotin on by magnetically separating it from the rest of the DNA that hasn’t got the biotin on. This leaves us with ssDNA.

dsDNA cannot form the secondary structures of interest.

Question 25

Define RNA interference (RNAi).

Answer 25

A biological process in which RNA molecules inhibit gene expression.

Question 26

Define small/short interfering/inhibitory RNA (siRNA).

Answer 26

A class of double-stranded RNAmolecules, 20-25 base pairs in length. siRNA is essentially a synthetic, man-made, miRNA molecule, and operates within the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, resulting in no translation.

Question 27

Define microRNA (miRNA).

Answer 27

A small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression.

Question 28

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment, such as small interfering RNA (siRNA) or microRNA (miRNA). The single strand acts as a ____1____ for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called ____2____, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes, and is a key process in gene ____3____ and defense against viral infections.

Answer 28

1. Template
2. Argonaute
3. Silencing

Question 29

What is the difference between siRNA and miRNA?

How does this alter functionality?

Answer 29

siRNA has perfect complimentary to target RNA, whereas miRNA binding is imperfect.

This causes siRNA to lead to the degradation of the target RNA, whereas miRNA just leads to a block in translation.

• Both prevent translation, but in different manners

Question 30

miRNAs and siRNA

Question 31

Define exogenous.

Answer 31

Growing or originating from outside an organism.

Question 32

Define endogenous.

Answer 32

Growing or originating from within an organism.

Question 33

Define transposon.

Answer 33

A chromosomal segment that can undergo transposition, especially a segment of bacterial DNA that can be translocated as a whole between chromosomal, phage, and plasmid DNA in the absence of a complementary sequence in the host DNA.

Question 34

Define transpose.

Answer 34

Cause (two or more things) to exchange places.

Question 35

miRNAs are transcribed by RNA ____1____ II and III.

Either independent genes or encoded within pre-mRNA introns.

Multi-miRNA precursor cleaved to produce pre-miRNAs in nucleus and transported to ____2____ for further processing and function.

Answer 35

1. Polymerase
2. Cytoplasm

Question 36

siRNA

• Part of the immune defence against ____1____ genetic elements
• Targets transposons and ____2____
• The target double stranded RNA is found in cytoplasm and is processed for function
• Some viruses have developed mechanisms for suppressing RNAi

Answer 36

1. Exogenous
2. Viruses

Question 37

What is Drosha?

What is Drosha’s function?

Answer 37

Drosha is a Class 2 ribonuclease III enzyme that in humans is encoded by the DROSHA.

The RNase III Drosha is the core nuclease that executes the initiation step of microRNA (miRNA) processing in the nucleus.

Question 38

Define argonaute protein function.

Answer 38

The Argonaute protein family plays a central role in RNA silencing processes, as essential catalytic components of the RNA-induced silencing complex (RISC).

Question 39

What is Dicer?

What is Dicer’s function?

Answer 39

An enzyme that in humans is encoded by the DICER1 gene.

Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively.

Question 40

Define exonuclease.

Answer 40

Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs.

Question 41

What is a processing body (P-body)?

Answer 41

Distinct foci within the cytoplasm of the eukaryotic cell consisting of many enzymes involved in mRNA turnover.

Question 42

Describe how we can use a virus as a machanism of siRNA delivery.

Answer 42

1. Add gene for siRNA to virus vector
2. Infect cells with virus
3. Virus will incorporate itself into the genome
4. Genome now contains a permanent copy of the synthetic siRNA

Question 43

Advantages and disadvantages of the different antisense approaches

Question 44

Name the methods of synthetic RNA delivery. (4)

Answer 44

• Conjugate to cholesterol
• Conjugate to an aptamer
• Envelop within liposome
• Complex with antibody and protamine