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BIOL 200 (5)

Post-transcriptional Control Textbook Notes

6 Pages

Biology (Sci)
Course Code
BIOL 200
Monique Zetka

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Start: page 345 Post-transcriptional Control Primary transcript: the initial mRNA right after transcription, aka pre-mRNA Steps: 5' structure added Splice out introns 3' end polyadenylated Co-transcriptional: pre-mRNA processing events (capping, splicing and polyadenylation) occur at the same time as the nascent mRNA is being transcribed Ribonucleoprotein (RNP) complexes: mRNAs are never free RNA molecules in the cell, but are associated with protein in RNP complexes First: nascent pre-mRNPs that are capped and spliced as they are transcribed Second: nuclear mRNPs after cleavage and polyadenylation Third: cytoplasmic mRNPs after export to the cytoplasm 5' Cap Why? 1. Protect the pre-mRNA from enzymes that digest uncapped mRNAs (5'-exoribonucleases) 2. Distinguish pre-mRNA from other types of RNA in the nucleus How? A cap is added to the 5' end after RNA Pol II makes 25 nucleotides - the cap is 7-methylguanosine and methylated riboses Catalyzed by a dimeric capping enzyme - enzyme associates with the phosphorylated CTD of RNA Pol II - only RNA Pol II has a CTD tail Capping enzyme removes y-phosphate from the 5' end of nascent mRNA Next the enzyme adds GMP to the 5' diphosphate end of transcript 1. Methyl group is added to N7 position of guanine 2. Methyl group is added to one or two 2' oxygens of the ribose In metazoans, the RNA is transcribed very slowly due to association of NELF Once the 5' cap is added, NELF is released by the CTD and transcription goes much faster Pre-mRNA: the intermediates of mRNA processing Heterogeneous Ribonucleoprotein particles (hnRNPs): contain hnRNA and proteins Heterogenous nuclear RNA (hnRNA): collective term for pre-mRNA and other nuclear RNAs hnRNP proteins contribute to splicing, polyadenylation and export through nuclear pores hnRNP Proteins - prevent pre-mRNA from base pairing complementary regions and forming short secondary structures (makes it inaccessible) - pre-mRNAs + hnRNP proteins make a uniform substrate for the next steps of processing - different hnRNP proteins prefer different regions of pre-mRNA Conserved RNA-Binding Motifs - RRM: RNA recognition motif, aka RNP motif, aka RNA-binding domain (RBD) = the most common binding domain in hnRNP proteins - 80-residue domain (present in other RNA-binding proteins) - has 2 highly conserved sequences (RNP1 and RNP2) - evolved early - RRM domain is 4 beta sheets next to 2 alpha helices - RNA phosphates are negatively charged, so the beta sheets make a positive surface - RNP1 and RNP2 lie next to the 2 central beta strands, and their side chains make lots of contacts with RNA that lies across the beta sheet - ex) KH motif (45 residue), found in hnRNP K protein, similar to RRM domain but interacts with RNA much differently - both are interspersed in 2 or more sets in a single RNA-binding protein - RGG box: an RNA-binding motif found in hnRNP proteins - has five Arg-Gly-Gly (RGG) repeats with interspersed aromatic amino acids RNA Splicing - Introns are removed, exons remain and are spliced together - Happens after polyadenylation of 3' end of short primary transcripts - Happens during transcription in long transcripts - splice sites = exon-intron junctions - Only certain portions of introns are necessary for splicing (first and last 30-40 nucleotides of an intron) - There are two transesterification reactions in splicing Intron Removal - removed as a lariat structure - 5' G of intron is joined in a 2',5' phosphodiester bond to an adenosine at 3' end of intron - This A residue is called branch point A because it forms an RNA branch in the lariat structure - 1 phosphodiester bond is exchanged for another in each transesterification reaction - no net change in # of phosphodiester bonds, so no energy is consumed - Net result: 2 exons are ligated & intron (branched lariat structure) is released snRNAs - snRNAs = small nuclear RNAs - base pair with the pre-mRNA and 170 associated proteins - 5 U rich snRNAs: U1, U2, U4, U5, U6 - participate in splicing - associated with 6-10 proteins each in snRNPs Spliceosomes = The five splicing snRNPs, other proteins, and pre-mRNA in a (ribonucleoprotein) complex - About as big as a ribosome Assembly: 1. a) U1 base pairs to 5' splice site b) SF1 protein (splicing factor 1) binds to branch point A c) U2AF (U2 associated factor) binds to the pyrimidine tract with large subunit, and to 3' AG with its small subunit 2. U2 base pairs with the branch point region as SF1 is released --> U1/U2/pre-mRNA complex 3. U4 and U6 snRNAs base pair a lot between each other and forms a complex that associated with the U5 snRNP = U4/U6/U5 "tri-snRNP" 4. "tri-snRNP" associates with the U1/U2/pre-mRNA complex 5. snRNAs and pre-RNAs rearrange, leading to the release of U1 snRNP 6. More rearrangement leads to the loss of U4 snRNP 7. This new complex catalyzes the first transesterification reaction - makes the 2',5' phosphodiester bond between 2' hydroxyl (on the branch point A) and the phosphate at 5' end of intron 8. Second transesterification reaction religates the two exons in a standard 3',5' phosphodiester bond - Intron is released as a lariat structure associated with the snRNPs - Intron-snRNP complex quickly falls apart and the snRNPs can start their work elsewhere, and the intron is degraded by a debranching enzyme and others Spliceosome - Has about 170 proteins, including 100 splicing factors in addition to the proteins associated with the five snRNPs - The process is very complex After RNA Splicing - some hnRNP proteins remain bound to the spliced RNA - they stay 20 nucleotides 5' to each exon-exon junction = exon-junction complex - RNA export factor (REF): one of the proteins associated with the exon-junction complex - exports the processed mRNPs from the nucleus to the cytoplasm - other proteins are quality control that degrade bad mRNA = nonsense-mediated decay How can you transcribe and process at the same time? - CTD has multiple repeats of a seven-residue sequence - it's very long - many proteins can associate with a single RNA pol II because of its length - the association of hnRNP proteins with the nascent RNA improves interaction of RNA pol II with elongation factors (DSIF, CDK9-cyclin T) to speed up transcription SR Proteins - exons are about 150 bases long, and introns are 3500 bases long - exons have the info defining the splice sites encoded in them - SR Proteins interact with exon sequences called exonic splicing enhancers - SR Proteins are a type of hnRNP proteins, so they have RRM RNA-binding domains - They have RS domains (rich in arginine and serine) - mediate the binding of U1 to the right 5' splice site and U2 to a branch point - cross-exon recognition complex = the complex of SR proteins, snRNPs and other splicing factors assembled across an exon - permit specification of exons in long pre-mRNAs Health Example - In organisms with long introns, exons have binding sites for SR proteins - Mutations interfering with SR protein binding to an exonic splicing enhancer prevent the formation of the cross-exon recognition complex - The exon is therefore skipping during splicing and is not in the final mRNA - If the mRNA is translated into a mutant, you can have such things as: - spinal muscular atrophy (mutation in SMN1 and SMN2 genes), SMN1 is inactivated - Sometimes the wrong site is recognized - Some mutations interfere with SR binding = exon skipping Trans-splicing: In protozoans, mRNA is made by splicing together separate RNA molecules Self-Splicing Introns - Some introns splice themselves
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