13 - RNA Splicing

Question 1

Introns key concepts

Answer 1

• Introns are removed from some transcripts
• this requires chemical interactions of a co-factor and chemical reactions of the products of this
• the events are coordinated by formation of base-paired structures in the RNA
• this structure holds the reactants in a locally concentrated manner
• this local high concentration promotes accurate splicing

Question 2

Bacterial vs eukaryotic transcription and translation

Answer 2

Bacteria:
- mRNA molecules translated whilst being transcribed
- generally not modified

Eukaryotic:
- mRNA precursors processed
- most spliced in the nucleus and transported to cytosol for translation

Question 3

Eukaryotic intron splicing visualisation

Answer 3

Question 4

Exon def

Answer 4

Any nucleotide sequence encoded by a gene that remains present within the final mature RNA product of that after introns have been removed by RNA splicing

Question 5

Intron def

Answer 5

Any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA produces of a gene is being regenerated

Question 6

Where can introns be found

Answer 6

Protein-coding genes (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)

Question 7

What RNA splicing involves

Answer 7

Removal of introns and covalent joining of exons to generate a mature mRNA or a mature non coding RNA product of a gene

Question 8

How intron possession variable in different organisms

Answer 8

• in higher eukaryotes, more DNA devoted to introns than to exons
• some of our genes have dozens of introns

Question 9

Discovery of split genes, 1977

Answer 9

Sharp and Roberts
- discovered using R-loop analysis

Question 10

What is R-loop analysis

Answer 10

RNA-DNA hybridisation can be monitored by electron microscopy, allowing analysis of gene organization, position and extension of homology regions, and characterization of transcription.

Question 11

R-loop analysis in bacteria and eukaryotes

Question 12

4 classes of introns

Answer 12

Group 1
Group 2
Spliceosome-dependent
Nuclear tRNA

Question 13

Group 1 intron info

Answer 13

• Self-splicing
• found in organelles (mitochondria, chloroplast)
• found in nuclear rRNA genes of some ciliates (unicellular eukaryotes)

Question 14

Group 2 intron info

Answer 14

Self-splicing (in organelles in fungi and plants)

Question 15

Spliceosome-dependent intron info

Answer 15

Found in nuclear mRNA

Question 16

Conserved features of introns

Answer 16

The 5’ splice site (start of intron)
3’ splice site (end of intron)
- both are absolutely conserved in all classes of introns to date

• branch site in spliceosomal and Class 2 introns are conserved

Question 17

Splicing of group 1 introns, 1982

Answer 17

• Thomas Cech
• purified rDNA of a bacteria
• added purified bacterial RNA polymerase
• but rRNA always spliced - why?

Question 18

Group 1 introns splicing info

Answer 18

• Group 1 introns can self-splice in the absence of any protein
• so RNAs have catalytic function - can be ribozymes
• done by two sequential transesterification reactions
• transesterification - process of exchanging organic R group of an ester with organic R group of an alcohol

Question 19

Splicing of group 1 introns - mechanism

Answer 19

• co-factor is required: guanosine, GMP, GDP, or GTP
• the 3’-OH of co-factor acts as a nucleophile that attacks phosphate at 5’ splice site
• 3’-OH of upstream exon becomes a nucleophile that attacks the phosphate at the 3’ splice site
• intron is ultimately degraded
• intron folds into tertiary structure
• results in 5’ and 3’ splice sites brought close together
• allows efficient and accurate transesterification reactions
• there is also a nucleotide binding pocket that presents the co-factor in the correct orientation

Question 20

Main difference between splicing of group 1 and 2 introns

Answer 20

• no co-factor required for group 2 introns
• instead, internal nucleophile is used
• 2’-OH of branch site adenine acts as a nucleophile and attacks phosphate at 5’ splice junction
• forms a lariat structure, and phosphodiester bond at 2’ and 5’

Question 21

Group 2 intron splicing mechanism

Answer 21

• no co-factor required for group 2 introns
• instead, internal nucleophile is used
• 2’-OH of branch site adenine acts as a nucleophile and attacks phosphate at 5’ splice junction
• forms a lariat structure, and phosphodiester bond at 2’ and 5’
• The 3’ -OH of the guanine of the upstream exon now acts as a nucleophile
• attacks the phosphate at the 3’ splice junction to complete the reaction.
• The result is fusion of the upstream and downstream exons and release of the intron in its lariat form.
• intron has a secondary structure determined by base-pairing rules, and then folds into a tertiary structure
• This results in the 5’ and 3’ splice sites and the branch site being brought close together
• allowing efficient and accurate transesterification reactions.

Question 22

Where group 1 introns found

Answer 22

• in the nuclear genomes of protists (in the rRNA genes)
• in rRNA, mRNA and tRNA genes of mitochondria in animals and fungi
• and in the tRNA genes and mRNAs of mitochondria and plastids in plants
• and are widespread in Archaea

Question 23

Where group 2 introns found

Answer 23

• in rRNA, tRNA, and mRNA of mitochondria in fungi and protists
• in rRNA, tRNA, and mRNA of mitochondria and plastids in plants
• and some have been found in Archaea

Question 24

2 hypotheses of intron origins

Answer 24

• intron-early hypothesis
• intron-late hypothesis

Question 25

Intron-early hypothesis info

Answer 25

since all three domains of life have introns, they must be of ancient origin - since modern organisms maintain them, they therefore must play a valuable role

Question 26

Intron-late hypothesis info

Answer 26

- some group 1 introns encode a homing endonuclease (HEG), which catalyses intron mobility - HEGs may move the intron from one location to another, and from one organism to another - thus these introns may be parasitic nucleic adids that encode a protein that allows them to spread selfiishly

Question 27

Spliceosome dependent intron splicing mechanism

Answer 27

- each snRNP (small nuclear ribonucleic particle) is a splicing factor - each snRNP comprises of - a snRNA (small nuclear RNA) - and at least seven protein subunits - the snRNPs associate to form an inactive spliceosome - base pairing of the U4 RNA with the U6 RNA inactivates U6 - the inactive spliceosome assembles - brings the splice sites closer together - U1 binds the 5' splice site, U2 binds the branch site - a preformed trimer of U4 5-6 bonds - dissociation of U4 snRNP activates U6 - this displaces U1 - forms an active spliceosome - the spliceosome provides a framework within which splicing occurs - the splicing reactions now take place within the spliceosome - the exons fuse and make mature RNA

Question 28

What is a snRNP - what are they made of

Answer 28

- small ribonucleic particle - each comprises of: - snRNA (small nuclear RNA) - at least seven protein subunits

Question 29

What are nuclear introns for

Answer 29

- used to be thought as junk DNA - no evidence for this - used for alternative splicing - all protein diversity - can be controlled developmentally - one gene can produce more than one protein

Question 30

How has spliceosome-mediated splicing evolved from group 2 intron splicing

Answer 30

spliceosome mediated intron splicing seems to have evolved from group 2 self-splicing - allows nuclear control of splicing and coordination of intron removal with transcription

Question 31

Exon shuffling info

Question 32

What can go wrong in intron splicing

Answer 32

- Mutations can destroy splice sites - leads to genetic diseases

Question 33

value of a complicated spliceosome

Answer 33

- improved efficiency of splicing - as base-pairing between U6 snRNA, U2 snRNA and the branch site causes the branch site adenine to sit on the bulge - brings it closer to the 5' splice site - so the first transesterification is more efficient - intron removal also becomes co-ordinated with transcription - rather than dependent on autocatalytic activity - so eukaryotic cell has taken control over the intron and its processes

Question 34

which intron splicing requires ATP, splicing endonuclease and a ligase enzyme

Answer 34

splicing with nuclear tRNA introns - have an independent mechanism - requires: - ATP - splicing endonuclease - ligase enzyme (cut and paste)

Question 35

transesterification def

Answer 35

process of exchanging organic R group of an ester with organic R group of an alcohol

Question 36

under what conditions can group 2 introns self-splice

Answer 36

high salt concentrations

Question 37

difference in splicing bewteen group 2 and spliceosome dependent introns

Answer 37

- catalytic process is identical to group 2 introns - catalytic RNA domains now encoded by splicing factors encoded by nuclear genes

Question 38

what process does spliceosome-mediated splicing seem to have evolved from? - advantages?

Answer 38

- evolved from group II intron self-splicing allows: - greater efficiency of intron removal - nuclear control of splicing - coordination of intron removal with transcription

Question 39

uses of nuclear introns

Answer 39

- alternative splicing - mechanism that generates protein diversity - can be controlled developmentally - exon shuffling