RNA Splicing Lecture Notes PDF

Lecture 13 – RNA splicing Part 1 – The discovery of split genes Eukaryotic genes are often split by introns 1. Gene turns into I° (primary) transcription through transcript 2. I° (primary) transcription processes into mature mRNA 3. Mature mRNA translated to protein Full Process of Gene Expre...

Lecture 13 – RNA splicing Part 1 – The discovery of split genes Eukaryotic genes are often split by introns 1. Gene turns into I° (primary) transcription through transcript 2. I° (primary) transcription processes into mature mRNA 3. Mature mRNA translated to protein Full Process of Gene Expression: Transcription: DNA → pre-mRNA (primary transcript) RNA processing: pre-mRNA → mature mRNA Translation: mature mRNA → protein 1. Transcription: The gene consists of a 5' UTR (untranslated region), three exons (coding regions), two introns (non-coding regions), and a 3' UTR. 2. RNA Processing: After transcription, the primary RNA transcript undergoes RNA splicing, where the introns are removed, and the exons are joined together to form the mature mRNA.  3. Translation: The mature mRNA is then transported to the ribosome, where it is translated into a protein, with the 5' UTR and 3' UTR playing regulatory roles in the process. From strand introns are removed and exons are joined by covalent bonds. This was discovered through R-loop analysis where mRNA hybridized with DNA template. R-loop analysis: mapping transcription to the genome Bacterial R-loop: A bacterial mRNA and a corresponding piece of genome DNA are mixed, heated to separate the DNA strands and cooled allowing hybridization. A single displaced loop of ssDNA indicates: - Where on the DNA the mRNA arose (gene mapping) - That the gene is contiguous (that is, no introns) The discovery of split genes – 1977 Sharp and Roberts independently analysed adenoviral genes and mRNAs by R-loop analysis There are multiple loops: mRNA base pairs with non-contiguous sequences of DNA. There are intervening sequences (introns) in the DNA that are not present in the mature mRNA Bacterial R-loop analysis  One displaced strands of ssDNA: no introns Two displaced strands of ssDNA (blue) and a loop of dsDNA (blue and orange): one intron Additional loops are seen if more than one intron is present RNA splicing: definitions Introns: any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of gene is being generated Exons: 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. Introns are found in a wide range of genes: - Protein-coding genes (mRNA) - Ribosomal RNA (rRNA) - Transfer RNA (tRNA) Part 2 – Group 1 introns and the hunt for a splicase  He did R-loop analysis in Tetrahymena He found RNA 17S and 26S The intron is excised from the primary transcript... When Tetrahymena nuclei were isolated and incubated with: 1.. α-amanitin (Pol II inhibitor) so no mRNAs could be made but rRNA genes were still transcribed 2. A nuclease inhibitor  3. And with ribonucleotides ATP, GTP, CTP and radioactive 32P-UTP 26S and 17S transcripts could be seen after gel electrophoresis and in strain 6UM there was free ~400b intron Cech worked out the mechanism of splicing: clue 1 (which conditions does splicase work?) In low salt conditions, there was minimal splicing observed. This means that salt conditions play a role in the splicing process. The image proposes that salts stabilize base-pairing, which allows the intron to form base-paired structures that bring the 5' and 3' ends of the intron together. This indicates that salt conditions influence the folding and structural organization of the intron, which is important for the splicing mechanism. The experimental data is presented as a gel electrophoresis image, where the "free intron" band suggests that under low salt conditions, the intron is not properly spliced out, remaining as a separate fragment. Clue 2: an extra G at the 5’ end of the intron The intron RNA sequence matched that of the DNA sequence except for an extra G residue on its 5’ end Clue 3: a G co-factor is required  Cech defined the minimum components necessary for release of the intron. Addition of GTP to transcripts purified afte low salt transcription stimulated splicing in vitro but addition of dGTP and ddGTP did not. When he added RNA and GTP, got splicases There is no splicase enzyme, so this experiment suggests that RNA has the ability to catalyse its own splicing reaction, which led to concept of ribozymes and the RNA world hypothesis. Cech worked out the 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. 2. 3. The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site. 4. The exons are fused, and the intron is ultimately degraded.  Two sequential transesterifications fuse the exons and release the intron 2 sequential transesterification reactions perform splicing Transesterification is the process of exchanging the organic group R” of an ester with organic group R’ of an alcohol Conclusion 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 and Some RNAs are ribozymes. Part 3 – A variation on a theme: Group II introns 4 known classes of intron 1. Group I introns: Self-splicing, found in organelles (mitochondria, chloroplasts) and in nuclear rRNA genes of some ciliates (unicellular eukaryotes such as Tetrahymena) 2. Group II introns: Self-splicing (in organelles in fungi and plants) 3. Spliceosome-dependent introns: Found in nuclear mRNA  4. Nuclear tRNA introns Conserved features of introns Conserved features: 1. 5’ splice site 2. 3’ splice site 3. branch site The 5’ splice site (at the start of the intron) and the 3’ splice site (at the end of the intron) are absolutely conserved in all classes of introns (to date). The branch site is found in Class II and spliceosomal introns Group II introns use an inbuilt co-factor 1. The intron folds and the 2’-OH of the ‘branch site’ adenosine attacks the phosphate at the 5’ splice site.  2. This adenosine now has three phosphodiester bonds: one is an unusual 2’, 5’ phosphodiester bond. The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site. 3. The exons are fused, and the intron is released as a lariat. Again, two sequential transesterifications fuse the exons and release the intron, this time as a lariat ORFs in introns: functions Group II introns can self-splice in vitro in high salt concentrations. In vivo, they require splicing factors, proteins that aid the splicing process. Group I introns can self-splice in vitro, in the absence of any other protein. However, a small number of group I introns are also found to encode maturases that improve the efficiency of intron splicing. And some Group I introns also encode a homing endonuclease (see later) Group I and Group II introns: found Group I and Group II introns: come from? Intron early hypothesis: 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: Some group I 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 acids that encode a protein that allows them to spread selfishly. Part 4 – Spliceosome dependent introns Spliceosome dependent introns (in nuclear mRNAs)  The catalytic process is identical to that Group II introns, but the catalytic RNA domains are now encoded by splicing factors that are encoded by nuclear genes Each splicing factor is snRNP (small nuclear ribonucleic particle Each splicing snRNP (U1, U2, U4, U5, and U6) comprises: - A snRNA (small nuclear RNA) - And at least seven protein subunits Spliceosome dependent splicing (1) U1 covers the 5’ splice site and U4 inactivates U6 by base-pairing. The important principle: an INACTIVE spliceosome assembles, bringing the splice close together. A helicase separates U4 and U6: U6 displaces U1: the 5’ splice site is exposed. The spliceosome is Activated. Spliceosome dependent splicing (2) The important principle: the spliceosome provides a framework within splicing occurs. Value of a complicated spliceosome Improved efficiency Base-pairing between U6 snRNA, U2 snRNA and the branch site causes the branch site adenine to sit on a bulge, bringing it closer to the 5’ splice site, making the first transestrification more efficient The phosphorylated C-terminal domain of the L’ subunit of RNA Pol II also recruits spliceosomes, so intron removal is coordinated with transcription. The eukaryotic cell has taken over control of the intron. Comparison between Nuclear tRNA vs Spliceosome-mediated splicing Nuclear tRNA introns have an independent splicing mechanism that requires ATP, a splicing endonuclease and a ligase (cut and paste). Spliceosome-mediated splicing appears to have evolved from Group II self-splicing and coordination of intron removal with transcription. Part 5 – what are introns for? What are nuclear introns for? Spliceosome-mediated splicing appears to have evolved from Group II self-splicing, allowing: 1. Greater efficiency of removal 2. Nuclear control of splicing and 3. Coordination of intron removal with transcription Rather than deleting introns from their DNA, eukaryotes appear to have evolved mechanisms for maintaining them in the genome. Why? The human dystrophin gene: - Encodes a protein of 11,055 base pairs - But the entire gene is 2.4 megabases long - It has 79 exons (coding regions), but 99.5% of the gene is introns (non-coding regions) - With a transcription rate of 40-50 nucleotides/second, it takes 13-17 hours to transcribe the entire g This is a big metabolic burden, so this suggests there are even more valuable advantages to maintaining nuclear introns than those listed above. Nuclear introns for: Alternative splicing Alternating splicing is a mechanism that generates protein diversity, and which can be controlled developmentally. The calcitonin gene can be used to generate 2 proteins products: calcitonin and calcitonin gene related peptide (CGRP) Nuclear introns: Exon shuffling