Lecture 13: RNA splicing Flashcards
Thursday 24th October
What is RNA splicing?
RNA splicing is the process of removing introns (non-coding regions) from a primary RNA transcript and fusing exons (coding regions) to produce a mature mRNA.
How was RNA splicing discovered?
By looking at R-loop analysis
Apart from coding sequences, what else do exons include?
UTR’s
What is R loop analysis?
Where a bacterial mRNA and a corresponding piece of genomic DNA are mixed, heated to separate the DNA strands, and cooled, allowing hybridisation. This is viewed under an electron microscope.
In R loop analysis of DNA, what does one strand of displaced ssDNA show in bacteria?
That there’s no introns
How is one intron shown through the R loop analysis of eukaryotes?
When there are 2 displaced strands of ssDNA and a loop of dsDNA. (Additional loops are seen if more than one intron is present.
)
What did Philip Shaw and Richard Roberts do in 1977?
They used adenovirus genes to show complex RNA-DNA loops, providing evidence for introns. They both won a nobel prize for their work on splicing.
What is an intron?
Any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of a gene is being generated.
What is an exon?
Any nucleotide sequence encoded by a gene that remains present within the final mature RNA product of that gene after introns have been removed by RNA splicing.
What types of genes are introns found in?
protein-coding genes (mRNA)
ribosomal RNA (rRNA) transfer RNA (tRNA)
Is it true that some introns are larger than our genes?
Yes
In many eukaryotes, is more DNA devoted to introns or to exons?
In many eukaryotes (including us), more DNA is devoted to introns than to exons: some of our genes have dozens of introns.
Describe Tetrahymena thermophila
Unicellular
Has a macronucleus and a micronucleus
Not all strains of Tetrahymena thermophila have an intron in the 26S rRNA gene.
Strain 6UM has a free intron
Thomas Kech
- While studying the ribosomal RNA of the unicellular organism Tetrahymena thermophila,
- Noted an intron through stuydng this organism, but he was not interested in it. This was an intron in the 26S rRNA gene and he noted that not all strains of Tetrahymena have this intron.
- To investigate the splicase, he isolated and incubated Tetrahymena nuclei with: α-amanitin (Pol II inhibitor) so no mRNAs could be made but rRNA genes were still transcribed; a nuclease inhibitor;
and with ribonucleotides ATP, GTP, CTP and radioactive 32P-UTP … - 26S and 17S transcripts could be seen after gel elctropheresis and in the strain with the intron, he found a 400base piece of RNA.
- Realsied that the intron is exercised from the primary transcript and that there should be a splicase that does this
How did Kech find the splicase
- He found that in low salt conditions, there was minimal splicing. Wondered if the introns could form intricate base pairs forming back on itself which would bring the 5’ and 3’ ends closer together, making it easier for the splicase to remove it.
- Realsied that the intron RNA sequence matched that of the DNA sequence except for an extra G residue on its 5’ end.
-Cech defined the minimum components necessary for release of the intron. Addition of GTP to transcripts purified after low salt transcription stimulated splicing in vitro but addition of dGTP and ddGTP did not. (Clues 2 and 3.
Was it a coincidence that GTP, the nucleotide that was found unexpectedly at the 5’ end of the intron was also the nucleotide required for intron release?
).
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Describe the ‘quiet’ experiment
Cech conducted an experiment adding only RNA, salts, and magnesium ions, along with a radioactive guanosine triphosphate (GTP).
The RNA spliced itself, proving that no protein enzymes [splicase] were necessary for the reaction.
He found that the RNA molecule folded into a specific structure that allowed it to catalyze the reaction by itself.
This process involved transesterification reactions, where the 3’-hydroxyl group of a guanosine acted as a nucleophile to break and reform RNA bonds.
Splicing mechanism
① The intron folds. A co-factor is held in a pocket: guanosine, GMP, GDP or GTP. The 3’-OH of the co-factor is a nucleophile that attacks the phosphate at the 5’ splice site. (This brings the 2 exons together).
② The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site.
③ The exons are fused, and the intron is ultimately degraded.
Is it true that the process of splicing is actually 2 sequential transesterification reactions?
Yes
Describe transesterification
Transesterification is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol.
Overall message
The Tetrahymena rDNA intron can self-splice in the absence of any protein as long as guanosine, GMP, GDP or GTP is present.
Thus RNAs can have catalytic functions –
Some RNAs are ribozymes.
How many known classes of introns are there?
4
Is it true that some RNA are ribozymes and so have catalytic functions?
Yes
What type of splicing do group 1 introns carry out?
Self splicing
Where are group 1 introns found?
In organelles (mitochondria, chloroplasts) and in nuclear rRNA genes of some ciliates (unicellular eukaryotes such as Tetrahymena)
