Lecture 20 - What happens to RNA between initiation of transcription and translation? Part 2 Flashcards
WWhat can be the result of alternative RNA splicing?
- Altered translation
- Altered protein structure
- Altered mRNA stability
What patterns are there of alternative splicing?
- Exon skipping
- Alternative 3’ splice site
- Alternative 5’ splice site
- Mutually exclusive exons
- Intron retention
What is the promary signal for determining whether the fly develops into a male or a female?
The X chromosome/autosome ratio
Female: 2 X chromosomes, 2 autosomes
Male: 1 X chromosome, 2 autosomes
What is mechanism involved in the Sxl section of the sex determination pathway in drosophila?
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
What are the features of RNA editing?
- 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
What are the two main ways that nucleotides can be modified in RNA editing
Deanimation
Cytidine -> Uracil
Adenosine -> Inosine (non stnadard base, read as guanosine in mRNA)
Why bother with nucleotide modification in RNA editing?
- amino acid change
- alternative splicing
- intron retention
- modulation of stability
- modulation of transport
Give an example of C to U editing
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
What is the mechanism of APOB C to U editing?
-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
What is mechanism involved in the transformer (2nd stage) section of the sex determination pathway in drosophila?
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)
What is mechanism involved in the doublesex (3rd stage) section of the sex determination pathway in drosophila?
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
What are the resulting forms of the Male and Female drosophila DSX proteins from the sex determination pathway (sxl, tra,dsx)?
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
How does the alternative splicing of drosophila Dscam RNA have the potential to produce a huge protein diversity from a single gene?
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
Give an example of developmentally regulated alternative splicing cascade
Alternative splcing of Drosophila Dscam RNA
-has the potential to produce huge protein diversity from a single gene
What is the Drosophila sex-determination pathway?
X chromosome/autosome ratio Sex-lethal (sxl) gene product Transformer (tra) gene product doublesex (dsx) gene product Male/Female fly
What are the features involved in the mutually exlusive exon inclusion in Dscam pre-mRNA?
- 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
Give an example of alternative splicing that shows the importance of RNA secondary structure (Slo gene in the nervous system)
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
What are the proteins involved in the ABOB C to U editing?
Transacting protein factors
-A cytidine deaminase APOBEC1 (APOB mRNA editing enzyme catalytic polypeptide 1)
-an auxillary factor, ACF (APOBEC1 complimetnation factor)
What is the tissue specificity of APOB editing determined by?
the expression pattern of APOBEC1
What are the features of C to U editing in plant organelles?
- 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
What are the features of A to I editiing?
- 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
Give some examples of A to I edited transcripts
- 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+
Why is high conductance of Ca2+ in uneditied AMPA receptors not favourable?
High conductance leads to severe epilepsy and death
How can RNA editing be involved in cancer?
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
How is RNA editing linked to environmental adaptation?
- 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
Why edit RNA?
-correct genomic errors, production of protein variety from single genes - NS especially prone to RNA editing
