L4 Posttranscriptional Regulation of Eukaryotic Gene Expression PDF

L4 Posttranscriptional regulation of eukaryotic gene expression. Explain the logic of gene regulation in eukaryotes. Describe and explain the molecular mechanisms underlying RNA splicing, editing and transport. Explain the role that alternative splicing plays in regulatin...

L4 Posttranscriptional regulation of eukaryotic gene expression. Explain the logic of gene regulation in eukaryotes. Describe and explain the molecular mechanisms underlying RNA splicing, editing and transport. Explain the role that alternative splicing plays in regulating gene expression. Describe how splicing modulates sex determination in drosophila Describe the diversity of biological functions of RNA-based mechanisms in health and disease. Outline modern experimental approaches used to investigate the posttranscriptional regulation of gene expression. Better understand the logic of gene regulation in eukaryotes: Eukaryotic gene expression unique because there is spatial separation of transcription in the nucleus and translation in the cytoplasm, providing opportunities for posttranscriptional regulation. Eukaryotes have a more complex gene structure with longer introns compared to prokaryotes, requiring additional specificity mechanisms and regulatory factors to ensure accurate splicing. The core cis-elements for splicing include: - 5' splice site - 3' splice site - Branch point sequence - Polypyrimidine tract  - The core trans-acting factors are the spliceosomal snRNPs and associated proteins - Regulatory RNA-binding proteins like SR proteins and hnRNPs can interact with these cis-elements and either promote or repress splicing depending on their binding position Processes subject to regulation: o Splicing o Editing o Transport It allows a single gene to produce multiple protein isoforms through alternative splicing, and for mRNA localization to play a key role in patterning and differentiation [19,31,32]. Improve your knowledge of molecular mechanisms underlying RNA splicing, editing and transport: Splicing Mechanisms: Pre-mRNA splicing is a key posttranscriptional process that removes introns and joins exons to produce the mature mRNA. It is the major mechanism for regulating gene expression. The spliceosome, a complex of snRNPs and associated proteins, catalyzes the splicing reaction Spliceosome consist of small nuclear RNAs, (snerps) (snRNPs) – U1,2,4,5,6. And other associated proteins. Steps of splicing o U1 snRNP binds to 5’ splice site. o Binding stabilised by protein factors. o Commitment complex formed from U1 and branch point binding protein and U2AF. (E complex). o U2 and ATP join and pair with branch site. o U4/6 and U5 (tri-snerp complex) join o U1 and U4 released. o Catalytic centre formed – U5 interacts with both exons through its loop. U6 pairs with 5’ splice site. o Transesterification reaction – 5’ splice site cleaved. o Second transesterification reaction – 3’ splice site cleaved. Additional RNA proteins involved in splicing regulation hnRNPs (heterogeneous nuclear ribonucleoprotein complexes) – bind introns and exons. Interact with splicing silencers. Tissue specific alternative splicing regulators – e.g. RBFOX, NOVA. Use Rna binding domains, KH domains and zinc fingers to bind to tissue specific  enhancers and silencers. How do RNA binding proteins affect splicing? For example: PTB affects spicing by: Competition with core splicing factors Assembly of higher order repressive complexes Inhibiting intron definition interactions Inhibiting exon definition interactions. The effect of RNA binding proteins on splicing decisions depends on their recruitment position relative to the exon. E.g. if it binds upstream it is inhibitory, if it binds downstream, it promotes splicing. E.g. binding to 5’ splice site increases U1 snRNP and leads to activation, whereas binding to 3’ splice site reduces U2 snRNP and causes repression. Transport Mechanisms Specific sequences and RNA-binding proteins can target mRNAs to distinct subcellular compartments, such as the budding tip in yeast or different regions of an oocyte, leading to important developmental outcomes and cell specialisation. For example, the localization of the ASH1 mRNA to the budding tip of yeast cells is crucial for establishing cell polarity and asymmetric cell division (endonuclease inhibitor). Editing Mechanisms In addition to splicing, eukaryotic mRNAs can also undergo editing, where specific nucleotides are chemically modified post-transcriptionally. The best-studied example is the conversion of adenosine (A) to inosine (I) by ADAR enzymes, which can alter the coding potential of the mRNA. Also, APOBEC1 can change C to U, which, if turned into UAA, is a stop codon. E.g. apolipoprotein B in liver = 4563 resides whereas in intestine has 2152 residues due to UAA codon being introduced. Appreciate the role that alternative splicing plays in regulating gene expression and appreciate the diversity of biological functions of RNA- based mechanisms in health and disease: Alternative splicing is a widespread mechanism in eukaryotes that allows a single gene to produce multiple mRNA isoforms Alternative splicing increases protein diversity and fine tunes gene expression, which controls mRNA abundance. Example 1: sex determination in drosophila The key genes involved are Sex-lethal (Sxl), Transformer (Tra), and Doublesex (Dsx). The initial trigger for sex determination in Drosophila is the ratio of X chromosomes to autosomes. XX chromosomes activate the early promoter of the Sxl gene, leading to the production of the Sxl protein The Sxl protein then acts in a positive feedback loop to maintain its own expression by controlling the alternative splicing of its own pre-mRNA. In females, Sxl promotes the inclusion of a female-specific exon, leading to the production of functional Sxl protein. In males, the absence of Sxl results in the inclusion of a male-specific exon containing a premature stop codon, leading to the production of a non- functional Sxl protein. In females, Sxl acts as a regulator on the Tra gene, so a functional Tra protein is produced. In males, no functional tra protein is produced. Tra proteins are SR proteins In females, tra acts as a splicing regulator, leading to the inclusion of the female doublesex exon. In males, the absence of tra results in the exclusion of the female doublesex exon, resulting in the male Dsx protein. Example 2: calmodulin kinase (splicing/transport) There is alternative splicing of exons in the calmodulin kinase gene to determine where the protein is localised to. Exons 13 and 17 always included Delta A: includes 13, 15, 16 and 17 – targeted to membrane. Delta B – contains 13, 14, and 17 – targeted to nucleus. Delta C – contains 13 and 17 only – targeted to cytoplasm. Example 3: iron response elements (condition specific mRNA stabilisation) In 3’UTR of transferrin receptor In low iron levels, IRE binding protein binds to IRE hairpins, which makes the transferrin receptor mRNA stable. In high iron levels, the IRE binding protein is released, meaning that the transferrin receptor mRNA is degraded.