Bio230 Lecture 6: Regulation Of Genome Expression - Fall 2024 PDF

BIO230 Section 1: Regulation of Genome Expression Lecture 6: Regulation of the Transcriptome BIO230H1F: From Genes to Organisms Prof. Kenneth W. Yip, Ph.D. Assistant Professor, Teaching Stream Cell & Systems Biology, University of...

BIO230 Section 1: Regulation of Genome Expression Lecture 6: Regulation of the Transcriptome BIO230H1F: From Genes to Organisms Prof. Kenneth W. Yip, Ph.D. Assistant Professor, Teaching Stream Cell & Systems Biology, University of Toronto Fall 2024 Hematopoiesis BIO230 Section 1 Lecture 6: Regulation of the Transcriptome Pg. 349-351 Mature Eukaryotic mRNAs are Selectively Exported from Post-transcriptional regulation the Nucleus From RNA to Protein RNA transport Pg. 358-361 An mRNA Sequence Is Decoded in Sets of Three mRNA quality control Nucleotides tRNA Molecules Match Amino Acids to Codons in mRNA mRNA stability Pg. 365-368 The RNA Message Is Decoded in Ribosomes Pg. 373-374 Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis Pg. 378-379 Quality-Control Mechanisms Act to Prevent Translation of Damaged mRNAs Readings (Alberts et al.): Pg. 462-466 Regulation of Gene Expression by Noncoding RNAs Small Noncoding Transcripts Regulate Many Animal and All listed on Quercus Plant Genes Through RNA Interference miRNAs Regulate mRNA Translation and Stability RNA Interference Also Serves as a Cell Defense Mechanism RNA Interference Can Direct Heterochromatin Formation Pg. 468-469 Bacteria Use Small Noncoding RNAs to Protect Themselves from Viruses BIO230 Lecture 1-6 2 A Review of RNA Processing Eukaryotic RNA processing is initiated before transcription is complete. How? BIO230 Lecture 1-6 3 A Review of the Coupling of Transcription and RNA Processing During transcription elongation, the C-terminal domain (CTD) of RNA polymerase binds RNA processing proteins and transfers them to RNA at the appropriate time The binding of RNA processing proteins is regulated by phosphorylation of RNA polymerase BIO230 Lecture 1-6 4 RNA Transport Out of the Nucleus The cell selectively transports mature mRNA from the nucleus Markers of mature mRNA must be acquired for export cap binding complex (CBC) exon junction complexes (EJC) poly-A-binding proteins These proteins travel with the mRNA to the cytosol Markers of immature mRNA must be lost for export proteins involved in RNA splicing (e.g., snRNPs) BIO230 Lecture 1-6 5 RNA Transport Out of the Nucleus The cell selectively transports mature mRNA from the nucleus. Only about 1/20 of RNA leaves the nucleus. Improperly processed mRNAs will eventually be degraded in the nucleus by the exosome. BIO230 Lecture 1-6 6 Regulation of Genome Expression Genome Transcriptome Proteome Post-Transcriptional Post-Translational DNA Transcription RNA Translation Protein Sorting Organization Splicing Localization Metabolome Interactome BIO230 Lecture 1-6 7 Post-Transcriptional Gene Regulation: mRNA Quality Control some mRNAs are incompletely processed or damaged in the cytosol need to prevent production of aberrant protein which can be toxic to cells First… Review of Translation tRNAs match amino acids to codons (3 nucleotides) in the mRNA genetic code BIO230 Lecture 1-6 8 Post-Transcriptional Gene Regulation: mRNA Quality Control Review of Translation mRNA message is decoded in ribosomes made up of >50 different proteins and several RNA molecules Amino acids are added to the C-terminal end of the growing polypeptide chain Therefore, proteins are Amino or synthesized from N-terminus N- to C-terminus Carboxy or C-terminus (COOH) BIO230 Lecture 1-6 9 mRNA Quality Control in Eukaryotes Translation initiation machinery recognizes the 5’-cap and poly-A tail: Eukaryotic initiation factors (eIFs) 5’ cap bound by eIF4E poly-A binding protein bound by eIF4G Recruit small ribosomal complex which will initiate translation at first AUG downstream of 5’ cap (some exceptions) Ensures that both ends of mRNA are intact The exon junction complex (EJC) also stimulates translation ensuring proper splicing… BIO230 Lecture 1-6 10 mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay prominent mRNA surveillance system surveys for nonsense (STOP) codons in the “wrong place” indicator of improper splicing BIO230 Lecture 1-6 11 mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay Normal Splicing The ribosome binds mRNA as it emerges from the nuclear pore EJCs are displaced by the moving ribosome The stop codon is in the last exon No EJCs remain bound when the ribosome reaches the stop codon mRNA is released in the cytosol BIO230 Lecture 1-6 12 mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay Abnormal Splicing The ribosome binds mRNA as it emerges from the nuclear pore EJCs are displaced by the moving ribosome The stop codon is premature The EJCs remain on the mRNA when the ribosome reaches the stop codon mRNA is degraded (mediated by BIO230 Lecture 1-6 Upf proteins) 13 mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay May have played an important role in the evolution of eukaryotes by allowing the selection of DNA rearrangements or alternative splicing patterns that produce full-length proteins Important role in cells of the immune systems where extensive DNA rearrangements occur to produce antibodies Also plays a role in many human disease cause by mutations that produce aberrant proteins. Cells can degrade aberrant mRNA and allow functional protein to accumulate BIO230 Lecture 1-6 14 mRNA Quality Control in Prokaryotes Prokaryotes also have quality control for incomplete or broken mRNAs Ribosomes stall on broken or incomplete mRNAs and do not release A special RNA tmRNA is recruited to the A site Carries an alanine amino acid Acts as both tRNA and mRNA BIO230 Lecture 1-6 15 mRNA Quality Control in Prokaryotes Broken mRNA is released Alanine is added onto the polypeptide from the tmRNA, which acts like a tRNA but with no anticodon-codon binding BIO230 Lecture 1-6 16 mRNA Quality Control in Prokaryotes The ribosome translates 10 codons from the tmRNA, which now acts as an mRNA The 11 amino acid tag is recognized by proteases that degrade the entire protein BIO230 Lecture 1-6 17 Post-Transcriptional Gene Regulation: mRNA Stability In prokaryotes, exonucleases rapidly degrade most mRNAs In eukaryotes, mRNAs are more stable and degradation is regulated… There are two main mechanisms: both involve gradual poly-A tail shortening Processes carried out by an exonuclease (deadenylase) when mRNA reaches the cytoplasm – acts as a timer of mRNA lifetime BIO230 Lecture 1-6 18 Post-Transcriptional Gene Regulation: mRNA Stability Once the poly-A tail reaches a critical length (humans = 25 nucleotides) two degradation mechanisms can occur both mechanisms can occur on the same mRNA cytoplasmic poly-A elongation can also occur to stabilize mRNA proteins can also interfere poly-A shortening (e.g., aconitase) BIO230 Lecture 1-6 19 Post-Transcriptional Gene Regulation: mRNA Stability and Transferrin Another example of protein-regulated mRNA stability… Transferrin Receptor imports iron into the cell needed when cellular iron is low mRNA stabilized by cytosolic aconitase binds 3’UTR aconitase binds iron and undergoes a conformational change mRNA released exposes 3’UTR endonucleolytic cleavage site (polyA removed); mRNA is degraded BIO230 Lecture 1-6 20 Post-Transcriptional Gene Regulation: mRNA Stability and Deadenylase Another example of protein-regulated mRNA stability… There is competition between mRNA translation and mRNA degradation deadenylase that shortens the poly-A tail binds the 5’ cap (like eIFs) BIO230 Lecture 1-6 21 Post-Transcriptional Gene Regulation: mRNA Stability and miRNAs non-coding RNAs called microRNAs (miRNAs) also regulate mRNA stability (>2000 in humans) miRNAs base-pair with specific mRNAs synthesized by RNA polymerase II and get a 5’ cap and poly-A tail after special processing, the miRNA associates with a protein complex called an RNA-induced silencing complex (RISC) BIO230 Lecture 1-6 22 Post-Transcriptional Gene Regulation: mRNA Stability and miRNAs RISC seeks mRNA with complementary nucleotide sequences A protein of RISC called Argonaute plays a critical role in base-pairing miRNA with mRNA Two possible outcomes are possible: BIO230 Lecture 1-6 23 Post-Transcriptional Gene Regulation: mRNA Stability and miRNAs RNA Interference (RNAi) Double-stranded RNAs that end up suppressing the gene expression of other RNAs in a sequence-specific manner The proteins used in the miRNA regulatory mechanisms also serve as a defense mechanism against foreign RNA molecules Found in eukaryotes, including fungi, plants, and worms BIO230 Lecture 1-6 24 Post-Transcriptional Gene Regulation: RNAi with siRNAs Many viruses (and transposable elements) produce double-stranded RNA as part of their life cycles RNAi destroys double stranded RNA Initiated by Dicer protein complex small interfering RNAs (siRNAs) siRNAs can interact with Argonaute and RISC proteins and follow the miRNA route to destroy double-stranded RNA or… BIO230 Lecture 1-6 25 Post-Transcriptional Gene Regulation: RNAi with siRNAs siRNAs can also regulate transcription siRNAs interact with Argonaute and the RNA-induced transcriptional silencing (RITS) complex RITS Interacts with newly transcribed RNA Recruits chromatin modifying enzymes BIO230 Lecture 1-6 26 Prokaryotic Immunity: CRISPR-Cas Immunity short fragments of viral DNA integrate into the CRISPR region of the genome and become templates to produce crRNAs (CRISPR RNAs) Viral DNAs complementary to CRISPR regions are directed for degradation by Cas (CRISPR-associated) proteins Similarly to Argonaute: use of small single-stranded RNA BIO230 Lecture 1-6 28 Prokaryotic Immunity: CRISPR-Cas Immunity short fragments of viral DNA integrate into the CRISPR region of the genome and become templates to produce crRNAs (CRISPR RNAs) Viral DNAs complementary to CRISPR regions are directed for degradation by Cas (CRISPR-associated) proteins Similarly to Argonaute: use of small single-stranded RNA BIO230 Lecture 1-6 29 CRISPR/Cas9 Applications Reprinted from "Applications of CRISPR/Cas9 for Genome Editing", by BioRender, May 2022, retrieved from https://app.biorender.com/biorender- templates/figures/5c8c7ba9d4f2ef3300632942/t-62547b825b6629012c87ef47-applications-of-crisprcas9-for-genome-editing Copyright 2022 by BioRender. BIO230 Lecture 1-6 30 Review Question 1 An mRNA is shown below. The intron indicated in purple is spliced out correctly. However, the 3’ intron is not spliced out. All other processing is performed correctly. What should be observed? A. An EJC will be removed from this mRNA. The EJC will mediate the degradation of this mRNA. B. There will be no EJC removed from this mRNA. There will be no EJC to mediate the degradation of this mRNA. C. There will be no EJC removed from this mRNA. The EJC will mediate the degradation of this mRNA. D. An EJC will be removed from this mRNA. There will be no EJC to mediate the degradation of this mRNA. BIO230 Lecture 1-6 31 Remember Review Question 2 From Lecture 1-5? A scientist places high levels of a miRNA targeting Sxl RNA into all cells of a developing Drosophila embryo. This results in Sxl RNA being specifically and completely destroyed. No other RNAs are destroyed. Which of the following will be observed? A. The Drosophila will develop into a male. B. The Drosophila will develop into a female. C. The Drosophila will develop into a male only if the X:A ratio is 0.5. D. The Drosophila will develop into a female only if the X:A ratio is 1. BIO230 Lecture 1-6 32 Remember Review Question 2 From Lecture 1-5? It can now be… A scientist places high levels of a miRNA targeting Sxl RNA into all cells of a developing Drosophila embryo. Which of the following will be observed? A. The Drosophila will develop into a male. B. The Drosophila will develop into a female. C. The Drosophila will develop into a male only if the X:A ratio is 0.5. D. The Drosophila will develop into a female only if the X:A ratio is 1. BIO230 Lecture 1-6 33 Review the textbook and add to your notes. If that was too quick for you, or if you have additional questions, please review the textbook, review the recording (if available), post on the Discussion Board, stay for the Q&A sessions, and try ChatBIO230. 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