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LF104: Molecules, Cells And Organisms > 13 - RNA Splicing > Flashcards

13 - RNA Splicing Flashcards

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1
Q

Introns key concepts

A
  • 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
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2
Q

Bacterial vs eukaryotic transcription and translation

A

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

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3
Q

Eukaryotic intron splicing visualisation

A
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4
Q

Exon def

A

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

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5
Q

Intron def

A

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

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6
Q

Where can introns be found

A

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

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7
Q

What RNA splicing involves

A

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

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8
Q

How intron possession variable in different organisms

A
  • in higher eukaryotes, more DNA devoted to introns than to exons
  • some of our genes have dozens of introns
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9
Q

Discovery of split genes, 1977

A

Sharp and Roberts
- discovered using R-loop analysis

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10
Q

What is R-loop analysis

A

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

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11
Q

R-loop analysis in bacteria and eukaryotes

A
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12
Q

4 classes of introns

A

Group 1
Group 2
Spliceosome-dependent
Nuclear tRNA

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13
Q

Group 1 intron info

A
  • Self-splicing
  • found in organelles (mitochondria, chloroplast)
  • found in nuclear rRNA genes of some ciliates (unicellular eukaryotes)
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14
Q

Group 2 intron info

A

Self-splicing (in organelles in fungi and plants)

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15
Q

Spliceosome-dependent intron info

A

Found in nuclear mRNA

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16
Q

Conserved features of introns

A

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
17
Q

Splicing of group 1 introns, 1982

A
  • Thomas Cech
  • purified rDNA of a bacteria
  • added purified bacterial RNA polymerase
  • but rRNA always spliced - why?
18
Q

Group 1 introns splicing info

A
  • 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
19
Q

Splicing of group 1 introns - mechanism

A
  • 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
20
Q

Main difference between splicing of group 1 and 2 introns

A
  • 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’
21
Q

Group 2 intron splicing mechanism

A
  • 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.
22
Q

Where group 1 introns found

A
  • 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
23
Q

Where group 2 introns found

A
  • 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
24
Q

2 hypotheses of intron origins

A
  • intron-early hypothesis
  • intron-late hypothesis
25
Q

Intron-early hypothesis info

A

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

26
Q

Intron-late hypothesis info

A
  • 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
27
Q

Spliceosome dependent intron splicing mechanism

A
  • 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
28
Q

What is a snRNP
- what are they made of

A
  • small ribonucleic particle
  • each comprises of:
  • snRNA (small nuclear RNA)
  • at least seven protein subunits
29
Q

What are nuclear introns for

A
  • 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
30
Q

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

A

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

31
Q

Exon shuffling info

A
32
Q

What can go wrong in intron splicing

A
  • Mutations can destroy splice sites
  • leads to genetic diseases
33
Q

value of a complicated spliceosome

A
  • 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
34
Q

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

A

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

35
Q

transesterification def

A

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

36
Q

under what conditions can group 2 introns self-splice

A

high salt concentrations

37
Q

difference in splicing bewteen group 2 and spliceosome dependent introns

A
  • catalytic process is identical to group 2 introns
  • catalytic RNA domains now encoded by splicing factors encoded by nuclear genes
38
Q

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

A
  • evolved from group II intron self-splicing
    allows:
  • greater efficiency of intron removal
  • nuclear control of splicing
  • coordination of intron removal with transcription
39
Q

uses of nuclear introns

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

LF104: Molecules, Cells And Organisms (25 decks)

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