BIO230 Lecture 5: Regulation Of Genome Expression PDF

BIO230 Section 1: Regulation of Genome Expression Lecture 5: 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 5: 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 5: Regulation of the Transcriptome 1. Applications of Transcriptome Analyses 2. RNA Processing Pg. 337-343 Transcription Elongation in Eukaryotes is Tightly Coupled to RNA Processing RNA Capping is the First Modification of Eukaryotic Pre-mRNAs RNA Splicing Removes Intron Sequences from Newly Transcribed Pre-mRNAs Readings (Alberts et al.): Nucleotide Sequences Signal Where Splicing Occurs All listed on Quercus RNA Splicing is Performed by the Spliceosome Pg. 348-349 RNA-processing Enzymes Generate the 3’ End of Eukaryotic mRNAs Pg. 446-448 Alternative RNA Splicing Can Produce Different Forms of a Protein from the Same Gene BIO230 Lecture 1-5 2 Various Techniques Allow for Transcriptome Analyses Analysis of a large set of cellular transcripts Provides a “signature” of cell type BIO230 Lecture 1-5 3 Transcriptome Analyses is Powerful! Provides a “signature” of cell state response to extracellular stimuli disease states e.g., cancer BIO230 Lecture 1-5 4 Transcriptome Analyses is Powerful! Provides a “signature” of cell state Primary acute myelogenous leukemia cells from patients Model cell line for AML Differentiated neutrophils Blue: low expression Red: high expression Stegmaier et al., 2004 BIO230 Lecture 1-5 5 Transcriptome Analyses is Powerful! Did it work?? IC50: Concentration of compound required to inhibit cell proliferation by 50% BIO230 Lecture 1-5 Stegmaier et al., 2004 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 1-5 7 A Review of RNA Processing Pre-mRNA Eukaryotic RNA processing is tightly coupled to transcription: 1. covalent modifications of RNA ends 2. removal of intron sequences BIO230 Lecture 1-5 8 A Review of RNA Processing: RNA Capping Addition of a modified guanine nucleotide to the 5’end of pre-mRNA (3 enzymes involved) Cap bound by cap-binding complex (CBC) Functions: 1. Helps in RNA processing and export from the nucleus 2. Important role in translation of mRNAs in the cytosol 3. Protects mRNA from degradation BIO230 Lecture 1-5 9 A Review of RNA Processing: RNA Splicing Recall that eukaryotic genes are made up of coding sequences called exons and non-coding sequences called introns Both are transcribed into RNA, but the introns are removed by a process called RNA splicing BIO230 Lecture 1-5 10 A Review of RNA Processing: RNA Splicing Different cells can splice an RNA transcript differently to make different proteins from the same gene: alternative splicing ~75% of human genes produce multiple proteins increases coding potential of genomes BIO230 Lecture 1-5 11 A Review of RNA Processing: RNA Splicing RNA splicing is carried out by an enzyme complex made up of RNA and proteins termed the spliceosome Sites of proper splicing are bound by exon junction complexes (EJCs) Serve as a marker for properly spliced RNA BIO230 Lecture 1-5 12 Alternative Splicing Can Be Regulated e.g., Drosophila sex determination BIO230 Lecture 1-5 13 Alternative Splicing Can Be Regulated: Drosophila Sex Determination ratio of X chromosomes (X): autosomal sets (A) X:A = 0.5 Male (Default) X:A = 1.0 Female Three genes involved: Sex-lethal: splicing repressor Transformer: splicing activator Doublesex: Regulates sex gene expression All 3 genes contain regulated splice sites BIO230 Lecture 1-5 14 Alternative Splicing Can Be Regulated: Drosophila Sex Determination Male, X:A = 0.5 Regulated Splicing? No Splice product is nonfunctional No Splice product is nonfunctional No Dsx protein represses female gene expression BIO230 Lecture 1-5 15 Alternative Splicing Can Be Regulated: Drosophila Sex Determination Female, X:A = 1.0 Regulated Splicing? 1st step: special Yes Sxl produced that Sxl protein represses is functional when splicing of Sxl and Tra spliced (not Yes shown) Tra protein activates splicing of Dsx transient Yes Dsx protein represses male gene expression BIO230 Lecture 1-5 16 Remember: Compare Male vs. Female Side-By-Side Don’t forget the details in your previous slides BIO230 Lecture 1-5 17 A Review of RNA Processing: 3’ Polyadenylation More complex than transcription termination in prokaryotes Signals encoded in genome RNA polymerase transfers protein complexes to RNA CstF (cleavage stimulating factor) CPSF (cleavage and polyadenylation specificity factor) BIO230 Lecture 1-5 18 A Review of RNA Processing: 3’ Polyadenylation and Termination RNA cleaved Transcription terminates Poly-A polymerase (PAP) adds ~200 A nucleotides to the 3’ end of RNA from ATP not genome encoded poly-A tail is bound by poly-A binding proteins aid in… - RNA export - translation - mRNA stability BIO230 Lecture 1-5 19 Review Question 1 Which of the following is typically true in Drosophila sex determination? A. In males, Tra RNA is not spliced and functional Tra protein is produced. B. In males, Tra RNA is not spliced and nonfunctional Tra protein is produced. C. In males, Tra RNA is spliced and functional Tra protein is produced. D. In males, Tra RNA is spliced and nonfunctional Tra protein is produced. BIO230 Lecture 1-5 20 Review Question 2 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 1-5 21 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. This is your responsibility.