Eukaryotic Transcriptional Regulation Past Paper PDF

Transcriptional regulation in eukaryotes 4 – Post- transcriptional modification Ben Nicholas Biol2010 2023-24 Semester 1 LO’s: At the end of this session you should be able to… List the key components of the spliceosome and how the help to regulate splicing eve...

Transcriptional regulation in eukaryotes 4 – Post- transcriptional modification Ben Nicholas Biol2010 2023-24 Semester 1 LO’s: At the end of this session you should be able to… List the key components of the spliceosome and how the help to regulate splicing events Describe the key processes and function of 3’ processing and polyadenylation of mRNA Outline how post-transcriptional processing enables efficient extra-nuclear transport of mRNA RNA rearrangements turn hnRNA (pre-mRNA) into mRNA RNA splicing oSelf splicing oSpliceosomal splicing oAlternative splicing RNA editing  Spliceosomal splicing is controlled and enabled by the spliceosome Co-transcriptional loading  RNAPol II carries with it many of the proteins involved in splicing  Much of the machinery of splicing assembles as the gene is being transcribed to form the spliceosome  Spliceosomes  Comprised of both RNA (5) and protein (150)  RNA component: small nuclear RNAs (snRNAs) U1 U2, U4, U5, U6 (100-200 nucleotides long)  Spliceosomes  Comprised of both RNA (5) and protein (150)  RNA component: small nuclear RNAs (snRNAs) U1 U2, U4, U5, U6 (100-200 nucleotides long)  Each RNA is complexed with 6-10 proteins to form small nuclear ribonuclear proteins (snRNPs)  Different complexes come in at different stages  Uses ATP  Spliceosomes  First U1 snRNP binds to 5’ splice site  U1 snRNP consists of U1 snRNA + proteins snRNP = small nuclear ribonuclear protein U1 small nuclear RNA 5’ end complementary to 5’ splice site Extensive secondary structure 1. Commitment/early complex U1 snRNP 5’ splice site BBP (SF1) branch point binding protein U2AF35 U2 auxiliary factors U2AF65 SR serine-arginine rich proteins snRNP = small nuclear ribonuclear protein 1. Commitment/early complex BBP U2AF65/35 U1 snRNP 5’ splice site BBP branch point U2AF35 3’ splice site U2AF65 polypyrimidine tract SR (serine-arginine rich) provide a framework - splicing enhancer 2. Pre-spliceosome/A complex BBP U2AF65/35 BBP U2AF65/35 U2 snRNP binds to branch site Aided by U2AF & displaces BBP Branch point A residue is excluded creating a single nucleotide bulge  3. Spliceosome is formed  U4,5 and 6 snRNPs now bind  The U2AF proteins are displaced  This brings the 5’splice site close to the branch point and 3’splice site  3. Spliceosome is formed  U4,5 and 6 snRNPs now bind  The U2AF proteins are displaced  This brings the 5’splice site close to the branch point and 3’splice site 2 cleavage reactions & joining of the exons  Self-splicing  Can occur in rare introns in which the intron codes for a ribozyme which splices itself out of the RNA  The ribozyme structure mimics the functions of the spliceosome without the additional proteins and shares similarity in reaction mechanisms  Hairpin and hammerhead structures  Could be considered the first parasitic elements.  Can re-organise elements of genes to produce more than one product from a limited RNA genome  Exon-exon shuffling can have an evolutionary effect. Alternative splicing can result in multiple products arising from the same gene  Alternative splicing  A process by which the RNA can be spliced in more than one way  This can result in the formation of 2 or more proteins from the same gene 5’ 1 2 3 4 5 6 3’  Alternative splicing  The 1st gene known to be alternatively spliced - Calcitonin/Calcitonin Gene Related Peptide (CGRP) Consists of 6 exons 5’ 1 2 3 4 5 6 3’ Thyroid 1 2 3 4 mRNA Protein Calcitonin Processed peptide  Alternative splicing  e.g. Calcitonin/Calcitonin Gene Related Peptide (CGRP) 5’ 1 2 3 4 5 6 3’ Thyroid Brain 1 2 3 4 mRNA 1 2 3 5 6 Protein Calcitonin Processed peptide CGRP  WHY?  Ordered and controlled process  Why does this happen in different cell types? - don’t yet understand!  It is clear that many of our genes are alternatively spliced  Examples of alternative splicing Oct-2 spliced into 12 different isoforms Pax-3 spliced into 5 different isoforms May explain why so few genes (25,000 instead of 100,000) 4. RNA editing  RNA editing  A process in which information changes at the level of mRNA Apolipoprotein-B has 29 exons CAA Codon 2153 codes for DNA glutamine Editing CAA UAA RNA In liver hepatocytes the In Intestine RNA codes for a 4563 a/a’s protein cholesterol and triglycerides  RNA editing Substitution editing Insertion/deletion  Cytidine deaminases editing convert a C in the RNA  Mediated by guide to uracil (U) RNA molecules  Adenosine  These base-pair with deaminases convert RNA an A to inosin (I) which  Serve as template for the ribosome addition/removal of translates as a G nucleotides  Both enzyme classes recognise target nucleotide sequence  RNA editing – Example 1  A process in which information changes at the level of mRNA Apolipoprotein-B has 29 exons CAA Codon 2153 codes for DNA glutamine Editing STOP!! CAA UAA RNA In liver hepatocytes the RNA In Intestine the RNA codes codes for a 4563 a/a’s protein for a 2153 a/a’s protein cholesterol and triglycerides dietary lipids  RNA editing  This is not a mistake - this is a specific controlled reaction Apolipoprotein-B has 29 exons CAA Codon 2153 codes for DNA glutamine Editing STOP!! CAA UAA RNA In liver hepatocytes the RNA In Intestine the RNA codes codes for a 4563 protein for a 2153aa protein  Editing reaction  This is a deaminase reaction in which the amino group on the nucleotide ring is removed Cytidine deaminase (RNA binding domain) 26 nucleotide region around CAA – targets enzyme  RNA Editing – Example 2  Also occurs in glutamate receptors - transmitter gated ion channel (in nervous system) - here there is an A to I (inosine) change  Editing reaction – Example 2  Also occurs in glutamate receptors - transmitter gated ion channel (in nervous system) - here there is an A to I (inosine) change - Catalysed by adenosine deaminase acting on RNA (ADAR) – there are 3 in humans  Editing reaction  Also occurs in glutamate receptors - transmitter gated ion channel (in nervous system) - here there is an A to I (inosine) change - Catalysed by adenosine deaminase acting on RNA (ADAR) – there are 3 in humans - acts on double stranded RNA AR - alters the Ca2+ AD permeability of the channel 3’  Editing reaction  Editing is vital - ADAR knock out mice: epilepsy and die A new function for the RNA-editing enzyme ADAR1 Hisashi Iizasa & Kazuko Nishikura ADAR1 catalyzes the deamination of adenosine to inosine in double- stranded RNA. This RNA-editing enzyme is now shown to be involved in hematopoiesis, where it acts to suppress interferon signaling and to block premature apoptosis. volume 10 number 1 january 2009 nature immunology  Summary  The main splicing events occur through the spliceosome  Additional splicing can occur through self-splicing events by ribozymes  Further PT changes can occur through RNA editing Lecture 5 – Preparation for transport to the ribosome  LO’s: By the end of this lecture you should be able to…  List the mechanisms and reasons for 3’ polyadenylation of mRNA  Outline how polyadenylation can go wrong and what the consequences are  Describe how the mature mRNA transcript is transported out of the nucleus  3. 3’ processing and  polyadenylation The 3’ end of the mRNA is polyadenylated  This involves the addition of a string of Adenosines (~200) to the end of the transcript  The signal for polyadenylation is encoded in the DNA (conserved 3’ control region)  Proteins bind to the poly A tail (PolyA Binding Proteins (PABP)) and help stabilise the RNA by resisting exonuclease attack 3’ processing and polyadenylation 10-30bp 10-20bp DNA 3’ AATAAA CA GT GT rich region 3’ processing and polyadenylation 10-30bp 10-20bp DNA 3’ AATAAA CA GT Everything gets GT rich region transcribed RNA 3’ AAUAAA CA GU  Sequences recognised by set of proteins DNA 3’ TGTA AATAAA CA GT CFI/II Poly A CstF RNA CPSF 3’ Pol UGUA AAUAAA CA GU CPSF = cleavage and polyadenylation specificity factor Poly A pol = Poly A polymerase CstF = cleavage stimulation factor CFI and CFII = cleavage factors I and II Components brought together by RNAPol II  The proteins work together to cleave the RNA CFI and Cleavage stimulation CFII cut factor CPSF Poly A CstF Pol RNA AAUAAA CA GU 3’ RNA AAUAAA CA  The proteins work together to cleave the RNA preRNA CPSF Poly A CstF Pol (hnRNA) AAUAAA CA GU 3’ Addition of a Poly A tail by Poly A Polymerase RNA AAUAAA CA A 200 approx 200 ribonucleotides mRNA AAAAAAAAAAAAAAAAAAAA Poly A binding proteins bind  Alternative polyadenylation Tian, B., Manley, J. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 18, 18–30 (2017). https://doi.org/10.1038/nrm.2016.116  Alternative polyadenylation BDNF – brain derived neurotrophic factor Transportation of the long form to the extremities of the cell is easier than transporting multiple proteins. As much protein as needed is made where it is required. Tian, B., Manley, J. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 18, 18–30 (2017). https://doi.org/10.1038/nrm.2016.116 Dysregulated alternative polyadenylation Gruber|&Zavolan, Nat Rev Genet 20, 599–614  5. Transport of RNA Only way to leave the nucleus is through the nuclear pore complex  Transport of RNA Only way to leave the nucleus is through the nuclear pore complex Small molecules can move through but larger molecules like RNA and proteins have to be transported across through an energy Exon junction complex (EJC) binds close to the dependent process splicing junction by association with the EJC spliceosome. EXON 1 EXON 2 After splicing the EJC remains attached to the RNA close to the exon/exon boundary. EJC proteins are targeted by Nuclear eXport Factor 1 (NXF1) and Nuclear eXport Transporter 1 (NXT1). NXF1 NXT1 EJC transports the RNA through EXON 1 EXON 2 the nuclear pore complex Ensures only fully spliced RNA transported NXF1 (Mex67) NXT1 (p15 or Mtr2) Gly-Phe (GF) peptide bound to NTF2L/p15 complex Katahira J. Nuclear export of messenger RNA. Genes (Basel). 2015 Mar 31;6(2):163-84. Nucleoporins or NUPs are GF rich proteins that line the NPC. Lodish 7th Edition, Fig 13-33 All mRNAs are transported through the NPC with the 5’ end going first. The proteins involved in transport are returned afterward back to the nucleus.  Capping Splicing Polyadenylation RNAP II CBC EJC NXF1/ NXT1 Part of the strategy of a virus is to force the invaded cell to focus on the production of the viral proteins. SARS-CoV-2 partly achieves this through the use of its Nsp1 protein which binds to the nuclear mRNA export machinery thus blocking fully formed mRNA in the nucleus from transfering into the cytoplasm. As the genome of SARS-CoV-2 is +ve strand RNA already in the cytoplasm, the virus is blocking competition for protein production by effectively removing the cellular mRNA from the pool of available mRNAs that are available to the ribosomes.  OVERVIEW OF EUKARYOTIC GENE EXPRESSION Post-transcriptional control adds layers of complexity to gene expression. Important in quality control of mRNA (only intact messages should be exported). Cap and tail help maintain stability. Recognition of these and e.g., EJC proteins, allows export from nucleus.  Summary  Polyadenylation stabilises the mRNA and helps to earmark for transport  mRNA transport to the cytoplasm from the nucleus occurs via the nucleopore complex  Pathogens can disrupt this process to boost their own cytoplasmic protein production  Overview  Transcription is a complex process involving the assembly of proteins into a machine that synthesises RNA from a DNA template  Involves initiation, elongation and termination, just like DNA and translation, but with specific differences related to RNA structure/function  Post-transcriptional events occur to regulate the production of mature mRNA from pre-mRNA and to ensure proper passage to the cytoplasm