L7 Control of eukaryotic translation.pdf

5BBG0205-Molecular basis of Gene expression Control of eukaryotic translation Dr Michelle Holland [email protected] Department of Medical and Molecular Genetics King’s College London Acknowledge...

5BBG0205-Molecular basis of Gene expression Control of eukaryotic translation Dr Michelle Holland [email protected] Department of Medical and Molecular Genetics King’s College London Acknowledgements: Sasi Conte Overall learning outcomes Understand the molecular mechanisms underlying translation initiation, elongation and termination To be able to describe how the stages of translation are regulated at a molecular level and to provide specific examples in different cellular contexts To describe the internal mRNA structures that contribute to translation regulation and in what context Objectives-Part 1 Review the core concepts surrounding translation Identify the primary components of the translation machinery: Ribosomes (rRNAs + proteins) Transfer RNAs (tRNAs) Messenger RNAs (mRNAs)  The central dogma (Crick) Transcription Translation DNA RNA Protein Reverse transcription Replication (e.g. HIV) “Deals with the detailed residue by residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid" Information transfer-from genes to proteins  Adaptor hypothesis (Crick) “each amino acid will combine chemically, at a special enzyme, with a small molecule which, having a specific hydrogen-bonding surface, would combine specifically with the nucleic acid template” The process of translation The genetic code Translation Ribosomes move from the 5’ to the 3’ end of the mRNA Amino acids are delivered on tRNA which possess the correct anticodon sequence (base pair complement) The ribosome catalyses the addition of the amino acid to the carboxyl end of the polypeptide The ribosome Mediates translation by catalysing the formation of the peptide bond between amino acids 2 subunits composed of proteins and RNA Small (40S) subunit “reads RNA” Large (60S) subunit “joins amino acids to form the peptide” Raza & Galili Nat Rev Cancer 2012 The ribosomal peptidyl transferase catalyses peptide bond formation Proposed mechanisms of ribosome activity Ribosomes enhance the rate of peptide bond formation by 107x by positioning the substrates correctly and excluding water from the active site The correct positioning of substrates is mainly accomplished by the 28S rRNA with ribosomal proteins L26 and L27 contributing Hydrogen bonding between the 2’-OH group of the P-site peptidyl tRNA (A76) and the attacking amino group is crucial to allow simultaneous extraction of a proton from the nucleophile and donation of a proton to the leaving group Transfer RNAs (tRNAs) The decode the codon sequence of mRNA and deliver the amino acid to the ribosome They have a “cloverleaf structure” Amino acids are attached o the 3’ end of the tRNA Transfer RNAs as the adaptors In the DNA/RNA information systems there are 43=64 possible codons Strict Watson-Crick base pairing between codon and anticodon would therefore require 64 unique amino acids However, we have less than 64 unique amino acids that occur naturally and contribute to proteins The wobble hypothesis The 3’ position in the codon within the mRNA permits non -Watson-Crick base pairing with the tRNA anticodon The allows a single tRNA to recognise multiple codons Reduces the number of required tRNA species Structure of a typical mRNA Internal ribosome entry site (IRES) Poly A tail (3’ end) Allows translation independently of Cap recognition 100-200 adenosines Effects mRNA stability 5’ untranslated region (5’UTR) 3’ UTR 5’ Methylated GTP ‘Cap’ UTRs – Untranslated regions Regulates nuclear export of mRNA mRNA stability Control mRNA stability & translation Critical for translation initiation efficiency (miRNAs) Binds eIF4E May contain IRES Objectives-Part 1 Review the core concepts surrounding translation Identify the primary components of the translation machinery: Ribosomes Transfer RNAs Messenger RNAs Objectives-Part 2 To be able to understand and explain the molecular mechanisms underlying the three key stages of translation: Initiation Elongation Termination To identify and describe the roles of the key subunits and factors that are involved in each stage of translation The key steps in translation initiation 40S ribosomal subunit associates with eIFs (43S complex) 43S complex is targeted to the 5’ end of mRNA (48S complex) Once bound, complex scans mRNA for AUG “start” codon 60S subunit associates, eIFs dissociate, translation begins  Translation initiation eIF1/1A and eIF3 bind to the 40S subunit eIF2/met-tRNA complex forms (GTP is an energy source) These two complexes assemble with tRNA-Met in the P site eIF5B-GTP binds (43S pre-initation complex) The mRNA 5’ cap is bound by eIF4E, this is then bound by eIF4G eIF4A acts as a helicase to remove secondary structures in the mRNA eIF4G binds to polyA-binding protein (PABP) to promote circularisation of the mRNA The 43S binds to the complex at the 5’ cap (48S complex) The 48S scans for the first AUG Upon recognition, GTP is hydrolysed and bound eIFs leave the 40S subunit The 60S subunit joins to form the 80S ribosome Albert et al., Molecular Biology of the cell Soluble factors involved in eukaryotic initiation Jackson et al., Nature Reviews Mol Cell Biol. 2010 doi:10.1038/nrm2838 Translation elongation through the open reading frame  Translation elongation Decoding tRNAs deliver amino acids to the ribosome Transpeptidation and “de-code” the codons of the mRNA The ribosome has 3 tRNA binding sites: A site: accepts the incoming aminoacyl Translocation tRNAs with then form a peptide bonds with the growing peptide chain in the P site. P site: holds the tRNA charged with the growing peptide chain E site: holds the tRNA that previously donated its amino acid Albert et al., Molecular Biology of the cell Ribosome contribution to translation fidelity The ribosome strongly prefers the correct tRNA The codon-anticodon base-pairing cannot account for the accuracy of tRNA selection (error rate of 10-3 to 10-4) The ribosome has a decoding sites that recognises the correct codon- anticodon interaction in the wider structural context, allowing for a high degree of translational fidelity  Peptide bond formation The ribosomal RNA acts like an enzyme TM Schmeing & V Ramakrishnan Nature (2009) doi:10.1038/nature08403 The energy requirements of translation GTP hydrolysis GTP hydrolysis The energy required to add the amino acid and Masaaki Sokabe, and Christopher S. Fraser Cold Spring move the ribosome is provided by the Harb Perspect Biol 2019;11:a032706 hydrolysis of GTP at several steps in reaction The role of elongation factors eEF1a: binds aminacyl-tRNA and GTP, recruits the aminoacyl-tRNA to the ribosome eEF1b: catalyses GDP to GTP on eEF1a eEF2: promotes translocation by binding to the A site of the 40S, displacing the tRNA after the peptidyl reaction Albert et al., Molecular Biology of the cell Translation termination at the stop codon Termination Occurs when a stop codon is reached (UAA, UAG, UGA) in the mRNA at the ribosomal A site Stop codons within this position are recognised by eRF1 Recognises stop codon with high specificity Extends into the peptidyl transferase centre to promote the release of the nascent peptide eRF3 hydrolyses GTP to provide the energy and is required for eRF1 and the ribosomal subunits to dissociate from the mRNA  Termination Occurs when a stop codon is reached (UAA, UAG, UGA) in the mRNA at the ribosomal A site Stop codons within this position are recognised by eRF1 Recognises stop codon with high specificity Extends into the peptidyl transferase centre to promote the release of the nascent peptide eRF3 hydrolyses GTP to provide the energy and is required for eRF1 and the ribosomal subunits to dissociate from the mRNA Albert et al., Molecular Biology of the cell Summary of the process of translation https://www.youtube.com/watch?v=qIwrhUrvX-k An mRNA may be translated by multiple ribosomes at once From: Young et al., (2016) J Biol Chem Albert et al., Molecular Biology of the cell Objectives-Part 2 To be able to understand and explain the molecular mechanisms underlying the three key stages of translation: Initiation Elongation Termination To identify and describe the roles of the key subunits and factors that are involved in each stage of translation Objectives-Part 3 To understand the main factors that can regulate the rate of cap- dependent translation initiation To be able to discuss specific examples of signalling pathways that converge on changing the rate of mRNA translation initiation To understand alternative methods of regulating mRNA translation initiation To understand regulation of translation elongation and termination Regulation of translation initiation The are two major complexes at which regulatory mechanisms act: 2. eIF2 eIF1A eIF3 eIF2 eIF4G 40S eIF4F eIF4E eIF4A complex 1. eIF4F eIF4F complex binding to mRNA eIF4E binds to the 5’ cap of the mRNA mRNAs are polyadenylated at the 3’ end eIF4G binds polyA binding protein (PABP) 5’ cap AAAAAAAAAA 4E 4G 40S P A B P Circularisation of the mRNA PABP binding to the mRNA polyA tail leads to circularisation of the mRNA 5’ cap 4E 4G 40S P A B P AAAAAAAAAA Circularisation enhances translation by allowing ribosome re-initiation by transfer of the 40S ribosome from the 3’ to 5’ end of the mRNA eIF4E regulation eIF4E is the rate limiting component of the eIF4F recruitment of the mRNA to the ribosome via the 5’ cap eIF4E regulation eIF4E-binding proteins (eIF4E-BP) bind to free eIF4E and compete with eIF4G for binding to eIF4E >> preventing initiation Growth factor/nutrient status regulates initiation through: Direct phosphorylation of eIF4E Phosphorylation of eIF4E-BP to prevent binding to eIF4E and promote association of the eIF4F complex with the 5’ cap.  eIF4E-BP phosphorylation in signalling In the absence or inhibition of mammalian target of rapamycin complex 1 (mTORC1) signalling (left), the eukaryotic initiation factor (eIF) 4E-binding protein 4E-BP is hypophosphorylated, which permits eIF4E binding and inhibits the formation of the eIF4F translation initiation complex. When mTORC1 signalling is active (right), mTOR phosphorylates 4E-BP; hyperphosphorylated 4E-BP cannot bind eIF4E, thus permitting eIF4F formation and the subsequent recruitment of eIF3 and the 40S small ribosomal subunit to the mRNA  eIF4E phosphorylation in signalling Cytoplasmic FMRP-interacting protein 1 (CYFIP1), in complex with fragile X mental retardation protein (FMRP), can bind eIF4E and inhibit the formation of the eIF4F translation initiation complex (left). Mitogen-activated protein kinase-interacting protein kinase (MNK) signalling stimulates the release of CYFIP1 from eIF4E (right), permitting formation of the eIF4F complex and recruitment of eIF3 and the 40S ribosomal subunit Bramham et al, Trends Biochem Sci 10:847 (2016) mTORC1 signalling Ma&Blenis, Nat Rev Mol Cell Biol 10:307 (2009) Regulation of translation initiation The are two major complexes at which regulatory mechanisms act: 2. eIF2 eIF1A eIF3 eIF2 eIF4G 40S eIF4F eIF4E eIF4A complex 1. eIF4F eIF2 regulation eIF2 needs the activity of eIF2B to exchange GDP for GTP to provide the energy for the complex to associate with the Met-tRNA Phosphorylation of the eIF2a subunit prevents the activity, inhibiting cap-dependent translation Holcok, Front. Oncol. (2015) eIF2a phosphorylation integrates the response to multiple stresses Bond et al, J Neuropath Exp Neurol 79:123 (2022)  Translational reprogramming in stress It limits resource usage and metabolic stress undertaken during normal cellular functions Reprograms translation of transcripts that respond to the stressors to ameliorate cell function Young&Wek, JBC (2016) Structure of a typical mRNA Internal ribosome entry site (IRES) Poly A tail (3’ end) Allows translation independently of Cap recognition 100-200 adenosines Effects mRNA stability 5’ untranslated region (5’UTR) 3’ UTR 5’ Methylated GTP ‘Cap’ Regulates nuclear export of mRNA UTRs – Untranslated regions mRNA stability Control mRNA stability & translation Critical for translation initiation efficiency (miRNAs) Binds eIF4E May contain IRES uORF regulation of GCN4 translation GCN4 is the yeast homologue of mammalian ATF4 Ternary complex (TC) availability is required for 40S ribosomes to scan for the next start codon allowing translation of additional upstream open reading frames (uORF) and promoting 40S dissociation prior to reading the start codon for the functional ORF. P-eIF2a limits TC availability and the scanning 40S scan further, improving the probability of reaching the correct start codon Zhang et al., Trends Biochm Sci (2019)  Cap-independent translation using an IRES Internal ribosome entry sites (IRES) are mRNA secondary structures. These can recruit the 40S ribosome subunit to the vicinity of the initiation codon without scanning from the cap May involve canonical eIFs or alternative IRES trans-acting factors (ITAFs) Lacerda etal, Cell Mol Life Sci (2017)  Viruses use IRES Translation of viral proteins is critical for replication Viral infection is a major trigger of the integrated stress response To favour translation of viral proteins over host proteins under these conditions, many viruses have IRES that can co-opt the host translational machinery when cap-dependent translation is inhibited Lee etal, Trends Microbiol 25:546 (2017) Regulation of translation elongation Phosphorylation of eEF1A and eEF1B enhance the rate of GEF activity, and increase the rate at Regulation of elongation through which eEF1A can rebind to an aa- tRNA following rare codons in response to each round of elongation - therefore increase the changes in cell stimuli may occur overall rate of aa-tRNA recruitment and by altering the concentration of translation. tRNAs that decode rare codons Phosphorylation of eEF2 by eEF2K (kinase) inhibits its activity by reducing its affinity for the ribosome, therefore Inhibiting elongation (and therefore translation) eEF2 phosphorylation therefore enables cells to survive nutrient deprivation Masaaki Sokabe, and Christopher S. Fraser Cold Spring Harb Perspect Biol 2019;11:a032706 - doi: 10.1101/cshperspect.a032706 eEF2 regulation of elongation rate Phosphorylation of eEF2 by eEF2K (kinase) inhibits elongation eEF2K is an atypical kinase (alpha kinase) activated by Ca2+ ions via calmodulin. It is also regulated by regulatory phosphorylation sites. Red or green denotes sites whose phosphorylation inhibits or activates eEF2K, respectively, which in turn activates or inhibits translation elongation respectively Kenney et al, 2014 http://dx.doi.org/10.1016/j.jbior.2014.04.003 Regulation of termination and recycling Masaaki Sokabe, and Christopher S. Fraser Cold Spring Harb Perspect Biol 2019;11:a032706 - doi: 10.1101/cshperspect.a032706 Translation targeting is toxic Objectives-Part 3 To understand the main factors that can regulate the rate of cap- dependent translation initiation To be able to discuss specific examples of signalling pathways that converge on changing the rate of mRNA translation initiation To understand alternative methods of regulating mRNA translation initiation To understand regulation of translation elongation and termination Objectives-Part 4 To understand the pathways of mRNA decay To be able to describe the contribution of the 3’-UTR to mRNA regulation Discuss the mechanisms by which non-coding RNAs (miRNA and siRNA) contribute to mRNA degradation  Deadenylation dependent mRNA decay mRNA stability directly influences the level of protein production Shortening of the polyA tail is initially reversible Can lead to degradation through two pathways  Translation initiation prevents deadenylation Factors that influence the rate of translation initiation for a particular transcript will also influence its stability Albert et al., Molecular Biology of the cell 3’ UTR sequences influence stability and localisation Mignone etal, Genome Biol 25:546 (2002) Mayya&Duchaine Front. Genet. (2019)  RNA interference Sequence-specific post-transcriptional gene silencing First described in worms (awarded Nobel in 2006) Albert et al., Molecular Biology of the cell  miRNAs in mammals Pri-miRNAs are cleaved by Drosha in the nucleus to give rise to pre- miRNAs Pre-miRNAs are cleaved to ~22nt dsRNA by Dicer This associates with the RISC complex and mediates the mRNA fate based on level of complementarity Albert et al., Molecular Biology of the cell  Cellular localisation in mRNA stability Processing bodies (P-bodies) are where RNA degradation takes place (in cytosol) mRNAs that are translationally repressed are moved to stress granules and may be translated again in the futre Albert et al., Molecular Biology of the cell Objectives-Part 4 To understand the pathways of mRNA decay To be able to describe the contribution of the 3’-UTR to mRNA regulation Discuss the mechanisms by which non-coding RNAs (miRNA and siRNA) contribute to mRNA degradation Overall learning outcomes Understand the molecular mechanisms underlying translation initiation, elongation and termination To be able to describe how the stages of translation are regulated at a molecular level and to provide specific examples in different cellular contexts To describe the internal mRNA structures that contribute to translation regulation and in what context Key resources Albert et al., Molecular Biology of the Cell, Garland https://pubmed.ncbi.nlm.nih.gov/15459663/ https://www.nature.com/articles/s12276-022-00757-5 Additional reading https://pubmed.ncbi.nlm.nih.gov/22888049/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954030/ https://pubmed.ncbi.nlm.nih.gov/31231778/ https://www.cell.com/trends/microbiology/pdf/S0966- 842X(17)30022-7.pdf The enzyme activity is RNA mediated (ribozyme) TM Schmeing & V Ramakrishnan Nature (2009) doi:10.1038/nature08403