13 - RNA Splicing Flashcards
Introns key concepts
- 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
Bacterial vs eukaryotic transcription and translation
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
Eukaryotic intron splicing visualisation
Exon def
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
Intron def
Any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA produces of a gene is being regenerated
Where can introns be found
Protein-coding genes (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
What RNA splicing involves
Removal of introns and covalent joining of exons to generate a mature mRNA or a mature non coding RNA product of a gene
How intron possession variable in different organisms
- in higher eukaryotes, more DNA devoted to introns than to exons
- some of our genes have dozens of introns
Discovery of split genes, 1977
Sharp and Roberts
- discovered using R-loop analysis
What is R-loop analysis
RNA-DNA hybridisation can be monitored by electron microscopy, allowing analysis of gene organization, position and extension of homology regions, and characterization of transcription.
R-loop analysis in bacteria and eukaryotes
4 classes of introns
Group 1
Group 2
Spliceosome-dependent
Nuclear tRNA
Group 1 intron info
- Self-splicing
- found in organelles (mitochondria, chloroplast)
- found in nuclear rRNA genes of some ciliates (unicellular eukaryotes)
Group 2 intron info
Self-splicing (in organelles in fungi and plants)
Spliceosome-dependent intron info
Found in nuclear mRNA
Conserved features of introns
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
Splicing of group 1 introns, 1982
- Thomas Cech
- purified rDNA of a bacteria
- added purified bacterial RNA polymerase
- but rRNA always spliced - why?
Group 1 introns splicing info
- 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
Splicing of group 1 introns - mechanism
- 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
Main difference between splicing of group 1 and 2 introns
- 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’
Group 2 intron splicing mechanism
- 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.
Where group 1 introns found
- 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
Where group 2 introns found
- 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
2 hypotheses of intron origins
- intron-early hypothesis
- intron-late hypothesis
Intron-early hypothesis info
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
Intron-late hypothesis info
- 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
Spliceosome dependent intron splicing mechanism
- 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
What is a snRNP
- what are they made of
- small ribonucleic particle
- each comprises of:
- snRNA (small nuclear RNA)
- at least seven protein subunits
What are nuclear introns for
- 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
How has spliceosome-mediated splicing evolved from group 2 intron splicing
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
Exon shuffling info
What can go wrong in intron splicing
- Mutations can destroy splice sites
- leads to genetic diseases
value of a complicated spliceosome
- 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
which intron splicing requires ATP, splicing endonuclease and a ligase enzyme
splicing with nuclear tRNA introns
- have an independent mechanism
- requires:
- ATP
- splicing endonuclease
- ligase enzyme (cut and paste)
transesterification def
process of exchanging organic R group of an ester with organic R group of an alcohol
under what conditions can group 2 introns self-splice
high salt concentrations
difference in splicing bewteen group 2 and spliceosome dependent introns
- catalytic process is identical to group 2 introns
- catalytic RNA domains now encoded by splicing factors encoded by nuclear genes
what process does spliceosome-mediated splicing seem to have evolved from?
- advantages?
- evolved from group II intron self-splicing
allows: - greater efficiency of intron removal
- nuclear control of splicing
- coordination of intron removal with transcription
uses of nuclear introns
- alternative splicing
- mechanism that generates protein diversity
- can be controlled developmentally
- exon shuffling
