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1

How do cis-splicing and trans-splicing of introns differ?

Cis-splicing is where the introns and exons are all part of the same transcribed region (or essentially part of the same gene)
Trans-splicing is where you can have two independent mRNA transcripts that are spliced together. They could come from completely different regions of the genome. Form a 'chimeric' transcript.

2

What sorts of proteins are involved in regulating intron splicing and how do they act?

Generally through regulatory proteins.
There can be negative or positive regulation.
Negative: a repressor at the 3' acceptor site prevents splicing of an intron
Positive: an activator can promote a poor/not normally cleaved site thus inducing intron splicing
Blocking a strong splice site might reveal a weak splice site
Known regulators are RNA-binding proteins

3

Describe the steps involved in formation of an intron lariat.

Intron removal is initiated by an adenine residue in the intron attacking the 5' splice site to form a looped intermediate.
The released 3' hydroxyl attaches to the start of the next exon, joining the two exons together and releasing the intron as a lariat (i.e. loop).
5' splice site = donor and 3' splice site = acceptor
Removal of nuclear gene introns is done by the spliceosome (snRNAs and protein).
The adenine is now linked to what was the 5' donor site (the GU) and has formed a phosphodiester bond forming a loop. A small stub trails off with the hydroxy group.

4

What is RNA editing? Describe two ways in which RNA editing can occur.

RNA editing is when you don't take things out (splicing) but instead alter the RNA itself. e.g. a substitution of one nucleotide for another (most common A to I, and C to U)
In trypanosome mitochondria: insertion of U nucleotides. RNA transcript is pair to Guide RNA (longer). Nucleotides in guide RNA specify the missing U nucleotides and editing occurs. Paired to Guide RNA 2 for final editing producing a fully edited mRNA. very clunky/ancient method. Doesn't happen as much in mammals.

5

What are exons and introns?

Exons are regions of a gene that are transcribed and retained in mRNA, introns are regions that are transcribed by not retained.

6

Why have RNA processing and how do we know it occurs?

RNA processing allows us to have a small(ish) number of genes (25-30000) but produce many more proteins and increase the complexity of the organism.
Dscam is a Drosophila gene involved in axon guidance: there are constitutive exons and there are 98 alternative exons aranged in 4 clusters (exons 4, 6, 9, and 17). As alternative exons are mutually exclusive (only 1 per cluster will be in the mRNA), Dscam can produce 38,016 different possible mRNAs, which lets every neuron in the fly display a unique set of Dscam proteins on its cell surface.

7

What are consesus sequences?

Sequences at either end of an intron and in the middle that are important for correct removal. GU at 5' end, AG at 3' end but otherwise there is a degree of flexibility

8

Are introns junk?

No: originally thought that introns were removed and degraded but this is no longer so. Introns can actually be functional as well.

9

How can alternative splicing be useful?

B cells in resting state have a membrane-bound antibody that recognise antigens.
When recognise foreign antigen they multiply and differentiate into effector B cells.
These now produce a secreted antibody that has the same specificity as the membrane bound antibody.
Cultured B cells that produce membrane-bound antibodies and secreted antibodies contain antibody mRNAs of two different lengths.
They found that the long RNA transcript (membrane bound) contained an intron that was removed. Translated protein had a hydrophobic AA at the C-terminal.
Short RNA transcript: pre-RNA is added earlier in the transcript. Intron sequence is not removed because acceptor splice junction is missing. Different sequence at C-terminal: hydrophilic. N-terminal is the same therefore same antigen specificity.

10

What are alternative ways of splicing an RNA?

Alternative 5' donor sites
Alternative 3' acceptor site
Exon skipping
Intron retention

11

Give an example of regulation of splicing

Sex determination in drosophila:
- determined by the ratio of X chromosomes to autosomes
- males are X:A= 0.5
- females are X:A = 1
- Sex-lethal gene (Sxl), Tra (transformer), and Dsx (doublesex) are then influenced by this ratio
- function in a cascade to determine sex
- male is default pathway:
- Sxl is nonfunctional in male due to splicing
- Tra splice combo produces a non-functional protein
- both these RNA transcripts contain male-specific exons that have stop codons producing non-functional proteins
- Dsx produces a protein in the male that represses female differentiation genes thus leading to male development (exon skipping during splicing)
- the Dsx protein contains 150 aa that are male specific (out of 550aa)
female:
- ratio changes start point of transcription of Sxl
- different promoter start point therefore different 5' end therefore different splice pattern
- splice site is blocked and the centre exon is skipped thus producing a functional Sxl protein
- Sxl protein is a negative regulator that binds its own transcript to block the splice site that is present in males as well as the splice site in Tra so another exon in skipped therefore forming functional Tra protein
-Sxl is a female specific repressor while Tra is a female specific activator that activates splicing to leave a female specific exon in Dsx
-Tra is a positive regulator of splicing: with Tra2 it binds Dsx and alters splicing pattern, retains the exon that was skipped in males
- Dsx protein has 30aa that are female specific - represses male differentiation genes leading to female development

12

give an example(s) of trans-splicing

The Drosophila mod(mdg4) gene is an example of trans splicing between two RNA molecules containing different coding regions.
The mod(mdg4) gene produces 26 different mRNAs that all contain common 5' exons and different 3' exons.
The 3' and 5' regions are transcribed independently and then spliced together.

In trypanosomes (parasitic protozoa) and nematodes (worms) trans-splicing occurs widely to give a Splice-Leader sequence. The SL RNA is independently transcribed and then spliced onto most of the mature RNAs in the cell, giving them a common 5' end. These leader RNAs are not translated.
Trans-splicing is catalysed by spliceosomes.

13

Give an example(s) of RNA editing.

Temperature adaptation in octopus neurons via RNA editing:
- in extremely cold water, K+ channels in neurons fire more slowly than in warm water. Octopuses at either pole use A-to-I RNA editing to produce channels that fire faster in the cold. Potassium channels in octopuses from temperate and tropical species have lower levels of A-to-I editing, in proportion to their water temperature.

C-to-U editing in apolipoprotein-b mRNA. In mammals, the apo-B gene is expressed in both hepatocytes (liver cells) and intestinal epithelial cells. In liver cells, its product is a 500kD protein called Apo-B100 and in intestine cells, its product is a small protein called Apo-B48. The Apo-B100 is produced without RNA editing. The Apo-B48 is synthesised from an mRNA where a CAA codon has been changed to a UAA stop codon.

14

Where do all the introns go?

Circular RNA.
Most eukaryotic genes contain multiple introns that occupy 90% or more of the protein-coding primary transcript.
Previously believed that intron lariats have no specific function and get degraded in the nucleus.
Now believed that circular RNAs are abundant, stable, conserved, non random and potentially functional.

15

How is it that circular RNAs mostly contain exon sequences?

Exon skipping can lead to a lariat containing exons that can be further spliced to form an exonic circular RNA.
The spliced exon sequences can form a loop, containing coding sequences that can bind other regions of RNA and inhibit its function.
This has been discovered due to advances in sequencing technology

16

What questions about RNA processing still remain?

- To what extent does alternative splicing account for organismal complexity?
- How many mRNA forms are functionally relevant?
- Is there a 'splicing code'? Can we predict where splicing will occur?