Lecture 20 - What happens to RNA between initiation of transcription and translation? Part 2 Flashcards

1
Q

WWhat can be the result of alternative RNA splicing?

A
  • Altered translation
  • Altered protein structure
  • Altered mRNA stability
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2
Q

What patterns are there of alternative splicing?

A
  1. Exon skipping
  2. Alternative 3’ splice site
  3. Alternative 5’ splice site
  4. Mutually exclusive exons
  5. Intron retention
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3
Q

What is the promary signal for determining whether the fly develops into a male or a female?

A

The X chromosome/autosome ratio
Female: 2 X chromosomes, 2 autosomes
Male: 1 X chromosome, 2 autosomes

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4
Q

What is mechanism involved in the Sxl section of the sex determination pathway in drosophila?

A

In males, the SXL transcript includes exon 3 that has a premature stop codon
-SPF45 is a splicing factor that binds to the AG dinucleotide and promotes the 2nd step of the splicing reaction with exon 3

In females, SXL (sex-lethal) regulates alternative splicing of it’s own pre-mRNA

  • SXL binds to a site adjacent to SPF45 and interferes with its activity as a splicing enhancer
  • exon 3 is therefore skipped

Sxl is involved in controlling splicing of the next gene in the cascade, transformer

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5
Q

What are the features of RNA editing?

A
  • first discovered in tryptanosome mitochondrial RNAs, involving the insertion and deletion of uridine nucleotides
  • modification of nucleotides is a very widespread type of RNA editing, observed in the nuclei of animal and plant organelles
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6
Q

What are the two main ways that nucleotides can be modified in RNA editing

A

Deanimation
Cytidine -> Uracil
Adenosine -> Inosine (non stnadard base, read as guanosine in mRNA)

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7
Q

Why bother with nucleotide modification in RNA editing?

A
  • amino acid change
  • alternative splicing
  • intron retention
  • modulation of stability
  • modulation of transport
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8
Q

Give an example of C to U editing

A

Apolipoprotein B (APOB) mRNA editing in mammals

  • APOB is crucial for transport of cholesterol and triglycerides in plasma
  • Two forms of APOB: long form in the liver (APOB100)
  • truncated form in the small intesting (APOB48)
  • in the small intesting, RNA editing causes a C to U conversion at position C6666
  • this results in a glutamine to stop codon change
  • very precise
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9
Q

What is the mechanism of APOB C to U editing?

A

-editing occurs with complete precision at C-6666
-requires both trans-acting factors and cis-acting sequence elements that surround the cytosine that is edited
Sequence elements ensure a favourable configuration for deanimation:
-Mooring sequence downstream of C-6666
-5’ and 3’ efficiency elements
-AU rich region
-Mooring sequence and 3’ efficiency element form a double stranded stem (if disrupted lose precision in editing)
-identified by site directed mutatgenesis

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10
Q

What is mechanism involved in the transformer (2nd stage) section of the sex determination pathway in drosophila?

A

In males, the proximal 3’ splice site of tra is used (promoted by U2AP)
-the mRNA produced contains a premature stop codon and the protein is therefore non-functional

In females, the Sxl product binds to the pre-mRNA and shift the binding of U2AF to a more distal 3’ splice site. A functional TRA protein is produces as the premature stop codon is skipped

Tra is then involved in controlling the splicing of the third gene in the cascade - Doublesex (dsx)

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11
Q

What is mechanism involved in the doublesex (3rd stage) section of the sex determination pathway in drosophila?

A

In males, the 3’ splice site of doublesex (dsx), immediately upstream of exon 4 is not recognised, thus exon 4 is skipped

In females, the TRA regulatory protein promotes the cooperative binding of an SR protein (RBP1) and an SR-like protein (TRA2) to exonic sequence enhancers (ESEs) within exon 4.

  • these splicing enhancer complexes then recruit the splicing machinery to the 3’ splice site
  • exon 4 has a polyadenylation site, meaning that the resultant protein will be truncated
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12
Q

What are the resulting forms of the Male and Female drosophila DSX proteins from the sex determination pathway (sxl, tra,dsx)?

A

Male DSX protein
-400aa joined to 150aa male specific C terminus
-repressed female differentiation genes
Female DSX protein
-400aa joined to a 30aa female specific C terminus
-repressed male differention genes

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13
Q

How does the alternative splicing of drosophila Dscam RNA have the potential to produce a huge protein diversity from a single gene?

A

12 variants of exon 4
48 variants of exon 6
33 variants of exon 9
2 variants of exon 17

more than 38000 possible Dsccam isoforms
-important in diversity and guiding axons

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14
Q

Give an example of developmentally regulated alternative splicing cascade

A

Alternative splcing of Drosophila Dscam RNA

-has the potential to produce huge protein diversity from a single gene

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15
Q

What is the Drosophila sex-determination pathway?

A
X chromosome/autosome ratio
Sex-lethal (sxl) gene product
Transformer (tra) gene product
doublesex (dsx) gene product
Male/Female fly
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16
Q

What are the features involved in the mutually exlusive exon inclusion in Dscam pre-mRNA?

A
  • upstream of each block of alternative exons is a docking site
  • upstream of each alternative exon is a selector sequence
  • normally splicing repressor proteins would be bound to the 3’ splice sites on each alternative exon
  • the docking sequence can base pair with one of the selector sequences, relieveing its repression

e.g. either between exon 5 and exon 7 there ccould be EITHER Exon 6.38 or exon 6.37 - mutually exclusive

17
Q

Give an example of alternative splicing that shows the importance of RNA secondary structure (Slo gene in the nervous system)

A

Slo gene from the nervous system is responsive to Ca2+

  • alters alternative splicing dependent upon Ca2+ concentration
  • SLO gene encodes a K+ channel
  • inclusion of the STREX exon makes the channel more sensitive to Ca2+
  • Ca2+ dependent protein kinase (CaMK IV) phosphorylates an unknown protein in the presence of high Ca2+ that supressed the inclusion of the STREX exon
18
Q

What are the proteins involved in the ABOB C to U editing?

A

Transacting protein factors
-A cytidine deaminase APOBEC1 (APOB mRNA editing enzyme catalytic polypeptide 1)

-an auxillary factor, ACF (APOBEC1 complimetnation factor)

19
Q

What is the tissue specificity of APOB editing determined by?

A

the expression pattern of APOBEC1

20
Q

What are the features of C to U editing in plant organelles?

A
  • changing amino acids and creating start or stop codons
  • precise but not 100% efficient
  • important for protein function (rather than diversity)
  • can rarely get U to C editing
21
Q

What are the features of A to I editiing?

A
  • the most common form of rNA editing in the nuclei of higher eukaryotes
  • relies upon 3 adenosine deanimases that act on RNA (ADARs):
  • ADAR1, ADAR2 and ADAR3 in mammals
  • common in the CNS
  • double stranded RNA is a common features of edited transcripts
22
Q

Give some examples of A to I edited transcripts

A
  • in mammals ~100% of transcripts of the GluA2 subunit of the AMPA receptor are edited to produce a glutamine to arginine change
  • crucial for correct function
  • AMPA receptors are glutamate activated cation channels mediating fast synaptic excitatory neurotransmission
  • receptors are assembled from four subunits
  • editied AMPA receptors are impermeable to Ca2+
23
Q

Why is high conductance of Ca2+ in uneditied AMPA receptors not favourable?

A

High conductance leads to severe epilepsy and death

24
Q

How can RNA editing be involved in cancer?

A

Heptidarcellular carcenoma

  • in normal cells the Antizyme protein targets oncoproteins CCND1 and ODC for degradation
  • increased RNA editing of AZIN1 mRNA results in a form of AZIN1 protein that sequesters Antizyme
  • LEvel of AZIN1 RNA editing increases with disease progresion
  • results in increased levels of CCND1 and ODC, increased cell proliferation and hepatocellular carcinome
25
Q

How is RNA editing linked to environmental adaptation?

A
  • closely related species can live in very different enviroments
  • e.g. polar v the tropical octopus
  • temperature sensitive neuronal synaptic transmission needs to adapt to the near freezing conditions of the polar octopus, (voltage gated K+ channel are highly temperature sensitive)
  • K+ channel in octopus DNA (tropical v. polar) shows no difference in sequence but difference in RNA
  • some editing events are specific to tropical/polar
  • editing at 1321V correlates with water temperature
26
Q

Why edit RNA?

A

-correct genomic errors, production of protein variety from single genes - NS especially prone to RNA editing