RNA Biology - Posttranscriptional Gene Regulation PDF

RNA Biology Posttranscriptional Gene Regulation André Gerber - Professor of RNA Biology, Dept. Microbial Sciences email: [email protected] • Lectures: 1) RNA processing in the nucleus Capping and Splicing Polyadenylation and mRNA export 2) Life and death of mRNAs in the cytoplasm mRNA localiza...

RNA Biology Posttranscriptional Gene Regulation André Gerber - Professor of RNA Biology, Dept. Microbial Sciences email: [email protected] • Lectures: 1) RNA processing in the nucleus Capping and Splicing Polyadenylation and mRNA export 2) Life and death of mRNAs in the cytoplasm mRNA localization Translation and decay 3) Non-coding RNA (ncRNA) Small ncRNA and rRNA Long non-coding RNA (lncRNA) • Tutorial: - Thrs 19 Oct 2023; 1 – 2 pm, LTD - Final revision: Fri 5. Jan 2024, 3 - 4 pm, LTD • Feedback: - Address question in the Discussion board - Test MCQ exam (& tutorials with example Qs) RNA Processing in the Nucleus (capping, splicing, polyadenylation) André Gerber PhD Professor of RNA Biology [email protected] BMS2036 Learning objectives • • • • • • You can name the pre-mRNA processing events occurring in the nucleus of eukaryotic cells You can explain the structure and function of the 7-methylguanosine cap at the 5’end of eukaryotic mRNAs You can outline the splicing mechanisms and you understand the role of snRNAs You know the impact of alternative splicing and how it can be regulated You can explain how the poly(A) tail is added at the 3’-end of mRNAs and understand how alternative polyadenylation site selection could be of physiological importance You know how eukaryotic mRNAs are exported to the cytoplasm Ø Lodish et al. Molecular Cell Biology, 8th ed. 2016, Chapter 10.1 Ø Alberts et al. Molecular Biology of the Cell, 7th ed. 2022, Ch6 From genes to protein in eukaryotes and prokaryotes exon intron Today’s lecture Recap: Structure of bacterial and eukaryotic mRNA molecules Operon 5’ UTR 3’UTR Figure 6-22a Molecular Biology of the Cell (© Garland Science 2022) Ø Supplement 1: Method for isolation of mRNAs from eukaryotic cells The carboxy-terminal domain (CTD) of RNA polymerase II acts as scaffold for assembly of RNA processing factors Pre-mRNA processing enzymes are recruited by the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. • • • 5’cap formation enzymes splicing proteins 3’-end processing factors Ø RNA processing starts cotranscriptionally RNA capping is the first modification of eukaryotic mRNAs • 7-methyl guanosine attached to the 5’-most nucleotide through an unusual 5’-P-P-P-5’ ester bond. • The 2’OH group of ribose 1 and 2 can be methylated (-CH3) • Rarely, the bases of residues 1 and 2 are also methylated Ø Supplement 2: Formation of the 5’cap in eukaryotic pre-mRNAs Figure 6-22b Molecular Biology of the Cell (© Garland Science 2022) Function of the 5’ cap in eukaryotic mRNAs Ø The 5’ cap marks RNA molecules as mRNA (other cellular RNAs do not have a cap!). • • • Nuclear cap binding proteins (CBPs) assemble at the cap and guide the nuclear export of mRNAs The cap protects mRNAs from RNA-digesting enzymes (5’exoribonucleases) In the cytoplasm, the cap promotes translation through interaction with a translation initiation factors (i.e., eIF4E, the cap-binding protein) Splicing: the removal of introns from the premRNA • 2’OH group of branch-site adenosine (A) attacks the phosphate group at the 5’ splicesite intron. 1. Transesterification • 3’OH group at 5’ splice-site attacks phosphate at 3’ splice site 2. Transesterification • Branched lariat is formed and rapidly degraded by nuclear exosome. Figure 6-26a Molecular Biology of the Cell (© Garland Science 2008) See also figure 8-8 Molecular Cell Biology, 7th ed. 2013 W.H. Freeman & Co. Consensus sequences define the splice sites in eukaryotic pre-mRNAs Cis-acting elements 5’ splice site invariant 3’ splice site “branch point A” (pre-mRNA) 20 – 50 b R=purine Y=pyrimidine Figure 6-28 Molecular Biology of the Cell (© Garland Science 2008) Recap lecture BMS1047 The “spliceosome” mediates pre-mRNA splicing Splicing requires: • • Combined molecular weight ~ 1.8 MDa • 5 small nuclear RNAs (snRNAs) U1, U2, U4, U5, U6 between 107 – 210 nts; each associated with proteins Total >100 proteins involved Hydrolysis of 8 ATP molecules to make RNA-RNA rearrangments How does the splicing machinery achieve specificity ? Fig.10-9 & 10-11 Molecular Cell Biology, 8th ed. 2016 W.H. Freeman & Co. Recap lecture BMS1047 Base-pairing between sequences in non-coding RNA (snRNAs) and “splice-site” sequences in the pre-mRNA define the splice-site Examples: 1) U1 snRNA::pre-mRNA annealing at the 5’ splice site 2) U2 snRNA::pre-mRNA annealing at branch-point U1 snRNA U2 snRNA only 6 bp!! Fig.10-9 & 10-11 Molecular Cell Biology, 8th ed. 2016 W.H. Freeman & Co. Model of spliceosome mediated splicing ATP>ADP ATP>ADP 1. U1 snRNP assembles at the 5’ splice site; U2 snRNP displaces the U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) at the branchpoint A. 2. A trimeric snRNP complex (U4, U5, U6) joins to form the spliceosome, bringing the 2 exons in close to each other 3. Rearrangement of basepairing interactions to from catalytically active spliceosome. U1/U4 snRNPs are released. Hydrolysis of 2xATP molecules! Model of spliceosome mediated splicing ATP>ADP ATP>ADP 4. Catalytic core catalyses the first transesterification reaction > intron lariat is formed. 5. Further rearrangement joins the two exons in a second transesterification reaction. 6. The excised lariat intron is converted to a linear RNA by a debranching enzyme and degraded by the exosome. https://www.youtube.com/watch?v=aVgwr0QpYNE#t=63 Exons are generally ~10 times shorter and more uniform than introns Size distribution of exons Size distribution of introns • Average length of human introns ~1,5 kb • Largest intron ~1,1 Mb (intron 5 in KCNIP4) Ø Since introns can be very long, additional strategies are required to improve splice site selection … • Average length of human exons ~150 bases • On average ~10 exons/gene Exon definition hypothesis: additional factors (SR proteins) help to guide snRNPs to splicesites • Serine-arginine-rich (SR) proteins often bind to exon sequences in the pre-mRNA (human SRSF1-SRSF12) • Heterogenous nuclear ribonucleoproteins (hnRNPs) often bind to intron sequences (~37 human hnRNPs) Alternative splicing in eukaryotes: generating mRNA variants from the same gene • Selection of alternative exons can generate transcripts variants which are translated into somewhat different protein isoforms • Alternative splicing can be highly cell/tissue-type specific • Besides the spliceosome – RNA-binding proteins can regulate the splicing of specific pre-mRNAs Wikipedia Ø “Diversity” can be increased with a relatively low number of genes! Recap lecture BMS1047 Example: The Drosophila DSCAM gene is the most extreme example of alternative splicing Down syndrome cell adhesion molecule (DSCAM) proteins have functions in i) neurons (neuronal cell-surface molecule to specify synaptic connections between neurons), ii) immune system (phagocytosis of pathogens) 12 48 Note: Drosophila has ~14,000 protein coding genes 33 2 One possible isoform Q: How many isoforms can be generated? Do the math: 12 x 48 x 33 x 2 = 38,016 possible isoforms !! Figure 7-57, Molecular Biology of the Cell, Garland 2015 Patterns of alternative splicing mRNA 1. mRNA intron exon (both mRNAs) 2. 3. 4. 5. exon (either mRNA) Control of alternative splicing by activator or repressor proteins Inhibition of splicing exon 1 intron exon 2 exon 1 exon 2 Repressorprevents Repressor hinders access access of of the spliceosome spliceosome Activation of Splicing Activator (splicing enhancer) enables splicing Ø Controlled by RNA-binding proteins (SR proteins >activators, hnRNPs >repressors) Figure 7-58, Molecular Biology of the Cell, Garland 2015 Mutation of splice-sites or in splicing factors can cause disease ! • ß-thalassemia o Inherited blood disorder (autosomal recessive) o Severe anemia due to abnormally low hemoglobin levels o Mutation of splice sites in the ß-globin gene (HBB) • Myotonic dystrophy o Neuromuscular disease, 2 types (DM1 and DM2) o DM1: Depletion of a splicing factor (MBNL) leads to missplicing of pre-mRNA targets. • Cystic fibrosis • Parkinson’s disease • Retinitis pigmentosa • Premature ageing • Cancer v see Table 10-21, Lodish et al. 6th ed. 2016 for further details Ø 10% of point mutations that lead to inherited human disease refer to aberrant splicing of the gene containing the mutation. Example: Splice site mutations in the human ßglobin HBB gene can cause ß-thalassemia Gel electrophoresis of reverse-transcription (RT) PCR products Watson: Gene cloning Recap: Cleavage and polyadenylation at 3’end of eukaryotic pre-mRNAs 3’end Pre-mRNA Poly(A) signal Downstream element mRNA • The 3’ end of eukaryotic mRNAs is NOT determined by the site of transcription stop, but rather by processing of the pre-mRNA. • Most eukaryotic mRNAs have 50-250 nts of adenosines (A) added to the 3’-end of their last exon – the poly(A) tail Recap lecture BMS1047 How does it work? Mechanisms of 3’ cleavage and polyadenylation Cleavage requires a protein complex consisting of: • CPSF (cleavage and polyadenylation specificity factor) • CstF (cleavage stimulatory factor) • Two cleavage factors (CFI, CFII) • Poly(A) polymerase (PAP). 1. CPSF binds to the AAUAAA sequence, CstF to the U-rich sequence, creating a loop. 1. 2. 2. PAP joins the complex; RNA is cleaved Figure 8-15 Molecular Cell Biology, 7th ed. 2013 W.H. Freeman & Co. Mechanisms of 3’ cleavage and polyadenylation 3. 3. CStF and CFs are released, and PAP adds ~ 10 As to the new 3’ end. 4. Poly(A) binding protein II (PABPII) binds to the short poly(A) tail and stimulates further addition of As 4. 5. 5. The whole poly(A) tail (~200 As) is ultimately covered by PABPII. What is the function of the poly(A) tail in eukaryotes? • Required for export of the mRNA from the nucleus to the cytoplasm (binding of PABPII) • Promotes translation initiation and translation • Stabilizes the mRNA > shortening of poly(A) tail may lead to reduced translation and eventual decay of the mRNA Ø Reminiscent to alternative splicing, selection of alternative polyadenylation sites can lead to different transcript variants in certain tissues/cells... Example: Regulated cleavage and polyadenylation determines whether antibodies are secreted or remain membrane-bound Intron Immunoglobulin H-chain Intron (low conc.) (high conc.) Intron CDS 3’-UTR (non-coding) CDS (…upstream 3’splice-site) 3’-UTR (non-coding) hydrophilic domain membrane domain protein B lymphocytes RNA export – how does the processed mRNA get out of the nucleus? Electron microscopy micrograph • mRNP Small molecules and proteins (<60 kDa) can diffuse trough the membrane • Macromolecular complexes (mRNPs) need active transport Remodeling of mRNPs during nuclear export Exchange of ‘common’ factors! -Nuclear capbinding protein (CBC) - Nuclear poly(A) binding protein (PABN1) Selectivity given by export factors/receptor Nuclear export factors (NXF1/T1) assemble with mRNA in the nucleus Nuclear pore complex (NPC) -Cytoplasmic capbinding protein (eIF4e) -PABPC1 NXF1/T1 are released in the cytoplasm and reimported to the nucleus Summary • • • • • • • • Pre-mRNAs are capped, polyadenylated, spliced in the nucleus (pre-mRNA processing). RNA processing factors are recruited co-transcriptionally to pre-mRNAs via phosphorylated CTD of Pol II. A 7-methyl-guanosine cap is added at the 5’-end of eukaryotic pre-mRNAs and protects the mRNA from degradation by nucleases. The spliceosome catalyzes two transesterification reactions that joins two exons and removes the intron as a lariat structure. A network of interactions between snRNPs and splicing factors determine splice sites selection. RNA-binding proteins bind to specific sequences near splice sites and regulate alternative splicing (SR proteins > activators, hnRNPs > repressors). Cleavage and polyadenylation at the 3’end of pre-mRNAs involves diverse RBPs and complexes. Alternative polyadenylation can have important physiological implications (e.g., antibodies). An mRNP exporter ensures directional export of mRNAs from nucleus to the cytoplasm through the NPC. The mRNP exporters are phosphorylated in the cytoplasm and release the mRNA cargo. Supplement 1: Isolation of mRNAs via oligo(dT) beads 1. Preparation of cell lysates under denaturing conditions 2. Capture of polyadenylated RNAs (mRNAs) by hybridization to oligo-(dT)25 coupled magnetic beads 3. Elute mRNAs from beads by heat Supplement 2: Addition of the 5’ cap 2. The gamma phosphate of residue 1 is removed. 3. GTP is added to the 5’ end of the mRNA in a 5’ - 5’ bond, with release of pyrophosphate (PPi). 4. Guanine base of cap is methylated on N7. 5. 2’OH of residues 1 and 2 are possibly methylated. Bases of residues 1 and 2 are rarely also methylated. Figure 10-3 Molecular Cell Biology, 8th ed. 2016 W.H. Freeman & Co.