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Genetics > Post transcriptional control of expression and RNAi > Flashcards

Post transcriptional control of expression and RNAi Flashcards

Alternative splicing, RNA editing, mRNA translation and stability, RNAi and miRNA mechanisms, uses of RNAi.

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Biology 101 flashcards
1
Q

What is an open reading frame (in eukaryotic mRNA)?

A

ORF - a continuous stretch of codons that may begin with a start codon (usually AUG but can be GUC/UUG) and ends at a stop codon (usually UAA/UAG/UGA).

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2
Q

What is present before the start codons and after stop codons in eukaryotic mRNA?

A

5’/3’ untranslated regions.

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3
Q

What is alternative splicing?

A

A process during gene expression that allows a single gene to produce multiple different mRNA molecules, leading to the synthesis of multiple protein variants.

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4
Q

What happens to the order of exons in alternative splicing?

A

Remains the same.

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5
Q

Give 4 types of alternative splicing.

(Look at summary sheet if confused).

A
  • alternative 5’ splice-site selection
  • alternative 3’ splice-site selection
  • exon inclusion/skipping
  • intron retention
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6
Q

What does tissue-specific alternative splicing of rat alpha tropomyosin RNA form?

A
  • striated muscle mRNA
  • smooth muscle mRNA
  • fibroblast mRNA
  • brain mRNA
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7
Q

Drosophila melanogaster (DSCAM) pre-mRNA has 95 alternative exons organised into clusters and forms 38,016 protein isoforms.

In the nervous system, what is DSCAM needed for?
In the immune system, what is DSCAM needed for?

A

Nervous system = wiring of neurones.
Immune system = phagocytosis of pathogens.

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8
Q

What is mutually exclusive splicing?

(If confused, look at PowerPoint)

A

One of exons in a cluster is included in functional transcripts.

Mutually exclusive splicing is a form of alternative splicing in which only one of several possible exons is included in the final mRNA transcript, while the others are excluded.

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9
Q

Alternative splicing is actively…

A

regulated.

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10
Q

What is negative control of alternative splicing?

A

Instead of the pre-mRNA transcript being spliced to mRNA, a repressor stops the splicing (so mRNA is the same as pre-mRNA).

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11
Q

What is positive control of alternative splicing?

A

Instead of the pre-mRNA transcript not being spliced to form mRNA, an activator retains its region and causes splicing of other regions without bound activators to form mRNA.

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12
Q

What is RNA editing?

A

Post-transcriptional editing of mRNA to further increase the number of distinct proteins that can be encoded in a genome.

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13
Q

Where is RNA editing a common phenomenon?

A

In parasites such as Trypanosoma brucei and Leishmania spp.
Also occurs in other organisms including animals.

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14
Q

After RNA editing, the mature mRNA sequence doesn’t correspond to the…

A

gene sequence.

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15
Q

What can RNA editing involve? (3)

A
  • addition or deletion of uridine to mRNA
  • A-to-I editing = deamination of adenine to
    produce inosine
  • C-to-U editing = deamination of cytosine to
    produce uracil

Deamination = the removal of an amino group.

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16
Q

Explain why Apo-lipoprotein B product is 4563 codons in the liver and 2152 codons in the intestine.

A

C-to-U editing of RNA generates UAA stop codon so protein in the intestine is a truncated version of 48 amino acids.

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17
Q

How do the roles of the protein in the liver and intestine differ?

A

Liver = major protein of plasma low-density lipoproteins (LDLs).

Intestine = Absorption of dietary fats.

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18
Q

How many human genes does RNA editing occur in?

A

~1000

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19
Q

Why is RNA editing done most of the time?

A

To make RNA stable.

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20
Q

Why is RNA editing done in parasites?

A

To make them resistant to attack from host organisms.

21
Q

Explain why the stability of an mRNA determines how much protein is translated in the cell.

A

mRNAs can be translated many times.

22
Q

What sequences are important in controlling mRNA lifetimes?

A

Untranslated regions.

23
Q

Why does iron need to be regulated in mammals?

A

It is an essential component of many proteins but can be toxic when circulating at high levels.

24
Q

What is the protein involved in iron uptake into the cell?

A

Transferrin receptor.

25
Q

What happens at low iron levels (transferrin mRNA)?

A

Iron response proteins bind to iron response elements at the 3’ UTR of transferrin receptor mRNA.

Transferrin receptor mRNA is stable so iron is transported.

26
Q

What happens at high iron levels?

A

Iron binds to iron response protein so these don’t bind to iron response elements on the 3’ UTR of transferrin receptor mRNA.

Transferrin receptor mRNA degraded so no iron transported.

27
Q

Information in UTR sequences can affect levels of…

A

translation.

28
Q

What is the role of ferritin?

A

Binds to iron to prevent it from becoming toxic.

29
Q

What happens at low iron levels (ferritin mRNA)?

A

Iron response protein binds to iron response element in 5’ UTR so translation is blocked and no ferritin is produced.

30
Q

What happens at high iron levels?

A

Excess iron is complexed. Iron binds to iron response protein so it can’t bind to iron response element in 5’ UTR so translation occurs and ferritin is produced.

31
Q

Which organism is used as a model for role of RNAs in controlling gene expression?

A

Caenorhabditis elegans = 0.5mm long nematode (round worm).

32
Q

Why are they good for these experiments?

A
  • cheap
  • easy to grow in Petri dishes
  • 3 day generation time
  • can be frozen
  • genetics: 2 sexes, mutants, hundreds of
    offspring, DNA and RNA can be introduced
  • developmental fate of all 959 cells known

Developmental fate = determines how many times a cell divides and final function in organism.

33
Q

What was the first animal to have its genome sequenced?

A

Caenorhabditis elegans

34
Q

In 1998, Andrew Fire and Craig Mello did work on RNAi in C. elegans, resulting in them winning the 2006 Nobel Prize in physiology/medicine.

What did they discover?

A

Injecting C. elegans with double stranded RNA reduced expression of specific genes by reducing levels of the mRNA.

This RNA interference is sequence-specific targeting of mRNA molecules.

35
Q

Give 2 roles of RNA interference in vivo.

A

Mediates resistance to both endogenous parasitic (e.g. transposable elements) and exogenous pathogenic nucleic acids (e.g. double stranded RNA viruses).

Regulates expression of protein-coding genes.

Endogenous = originating/growing from inside organism.

36
Q

Give the mechanism of RNAi.

(Look at summary sheet if confused).

A
  1. Foreign double stranded RNA is cleaved
    by dicer into double stranded siRNAs.
  2. Formation of RISC (RNA-induced silencing
    complex).
  3. Forms single stranded siRNA which
    searches for complementary RNA.
  4. siRNA (and RISC) forms complementary
    base pairs with single stranded foreign
    RNA.
  5. RISC released and foreign RNA degraded.
37
Q

Give the mechanism for micro RNAs.

(Look at summary sheet if confused).

A
  1. Precursor (pre) miRNA is processed and
    exported from nucleus to cytoplasm into a
    double stranded RNA intermediate.
  2. Formation of RISC.
  3. Forms single stranded miRNA which
    searches for complementary target mRNA.
  4. RISC and ss miRNA form an extensive
    match with mRNA and mRNA is rapidly
    degraded by nuclease within RISC.
    OR
  5. RISC and ss miRNA form a less extensive
    match with mRNA so translation is
    reduced because mRNA is sequestered
    and eventually degraded by nucleases in
    cytosol.
  6. RISC released.

Sequestered = isolated/hidden.
Cytosol = aqueous component of cytoplasm which various organelles and particles are suspended in.

38
Q

When were miRNAs discovered?

A

Relatively recently.

39
Q

What is the role of miRNAs?

A

Key regulators of gene expression in many biological processes.

40
Q

Why is RNAi used as an experimental tool for gene ‘knockdown’?

A

It can be used to temporarily stop expression of 1 or more targeted genes.

41
Q

Describe how RNAi is used for gene knockdown in C. elegans.

A
  1. E.coli expressing double-stranded RNA is
    eaten by worm.
  2. Double-stranded RNA is injected into gut.
  3. Double-stranded RNA is processed into
    interfering RNA bound to Argonaute or
    Piwi proteins.
  4. Target RNA binds to interfering RNA by
    complementary base pairing.
  5. Cleavage of target RNA.
    OR
  6. Translational repression and eventual
    destruction of target RNA.
    OR
  7. Formation of heterochromatin on DNA
    from which target RNA is being
    transcribed.
42
Q

What happens in an embryo in which a particular gene (coding for a kinesin called ceMCAK) has been inactivated by RNAi?

A

The pronuclei fail to migrate.

Pronuclei = nuclei found in sperm and egg cells during process of fertilisation.

Kinesin is a motor protein that plays a crucial role in cellular transport.

43
Q

Give 3 uses of RNAi.

A

As an experimental tool for genome-wide genetic screens.
Tool to control infectious disease.
In medicine to treat transthyretin-mediated amyloidosis.

44
Q

Experimental tool for genome-wide genetic screens:

Each well in a 96 well plate contains E.coli expressing a different ds RNA and C. elegans are added.
What happens next?

A

Worms ingest E.coli.
Resulting phenotypes are recorded and analysed.
Wildtype vs Sterile C. elegans.

45
Q

dsRNA can potentially be used to control viral diseases in farmed shrimp.
Explain how dsRNA can be administered.

A

Orally by dsRNA-enriched bacteria/artemia (brine shrimp/sea monkeys).

OR

Intramuscular administration in non-biological/biological nanocontainers.

46
Q

How does transthyretin-mediated amyloidosis make a person ill?

A

TTR (transthyretin protein in cells that circulate in blood) synthesised by liver (from TTR mRNA) becomes folded dimers, folded monomers, then misfolded amyloidgenic monomers which form small oligomers and amorphous aggregates (mis-folded protein clump). This forms amyloid fibrils (resistant to degradation).

Results in restrictive cardiomyopathy (walls of heart chambers become stretched/thickened/stiff).

AND

Autonomic neuropathy (damage to nerves controlling automatic body functions).

AND

Peripheral neuropathy (affects nerves outside brain and spinal cord).

The misfolding of transthyretin (TTR) proteins is primarily due to mutations in the TTR gene.

47
Q
A
48
Q

A patient receives onpattro directly into bloodstream via vein to control increased expression of TTR mRNA.
How does this result in decreased/stabilised cardiomyopathy and neuropathy?
Give description of mechanism.

A
  1. Onpattro contains ds siRNA (small
    interfering RNA). siRNA binds to APOE
    protein on a lipoprotein particle and binds
    to APOE receptor (on hepatocyte = liver
    cell).
  2. siRNA is contained in an exocytic vesicle
    in cell.
  3. ds siRNA is released and cleaved by dicer
    to form guide strand.
  4. Guide strand binds to RISC which binds
    to TTR mRNA.
  5. mRNA cleaved so less TTR protein
    (mutated/wild type) produced.

Genetics (10 decks)

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