BMSC 320 Nucleic Acids: From Central Dogma to Human Disease PDF

BMSC 320 Nucleic Acids: From Central Dogma to Human Disease Asynchronous Lecture 27 November 25, 2024 Why does the mismatch matter? Fully complementary siRNA mimics some viral infections that use dsRNA, so degradation evolved as a defense mechanism Mismatched miRNA can acco...

BMSC 320 Nucleic Acids: From Central Dogma to Human Disease Asynchronous Lecture 27 November 25, 2024 Why does the mismatch matter? Fully complementary siRNA mimics some viral infections that use dsRNA, so degradation evolved as a defense mechanism Mismatched miRNA can accommodate many more targets and be used as an additional control mechanism to downregulate translation of multiple genes simultaneously Extent of miRNA/mRNA Binding Varies Typically the “seed” region at the 5’ end of the miRNA matches 7-8 bases of the target mRNA Usually in the 3’ UTR Less likely – 5’ UTR or coding region Other miRNAs may bind by looping out mismatched portions The extent of the match determines the strength of the interaction & silencing miRNAs and mRNAs are “tuned” over time to give optimal gene expression levels  blue = known red = known & predicted green = predicted This 2010 paper looked at 56 species for highly conserved miRNAs and found some span worms to fish to chickens to humans Example: miRNA in muscle development miR-1 is required for muscle development Deletion of miR-1-1 in mice leads to lethality and heart defects Example: miRNA in muscle development Known miRNAs involved in muscle development and the processes they regulate Simpler Muscle Development Skeletal muscle growth and regeneration is a delicate interplay between: miRNAs transcription factors (Pax3, Pax7, SRF) Signals & cascade proteins (IGF-1, PTEN) Histone deacetylases (not shown, but silenced by mir-1) Others (myomaker is a membrane protein allowing myoblast fusions) Example: miRNAs in tissue damage & repair miR-1 & miR-133 regulate heart growth, differentiation and even modify cardiac rhythm Research is showing some miRNA levels in blood show a high degree of change after heart attacks Altered expression after heart attack is an indicator of the extent of damage & cellular repair processes & time since damage Research is being done to use miRNA levels for diagnostics for infections, liver disease, various cancers, endometriosis  up-miRNA? Although there are only limited experiments to date, multiple models/organisms show miRNAs can also enhance translation Example: FXR1 is an RNA binding protein that also associates with the large ribosomal subunit upregulation of translation likely due to more efficient recruiting of ribosomes The question remains – What gets recognized by FXR1 to know which miRNAs are up-miRNAs? The Power of A Good Reporter System Eukaryotic plasmid is transformed into mammalian cells Plasmid contains dual fluorophores expressed at a consistent ratio Target gene’s 3’ UTR is cloned downstream of cerulean Cells are treated with a library of miRNAs Ratio of blue/red is measured Results here: the ST6GAL1 3’ UTR shows regulation by only 4% of the miRNA library, but the effector miRNA’s are 3X more likely to upregulate than downregulate Another regulator: circRNAs RNA can be spliced in a way that produces an RNA with no 5’ or 3’ ends as it’s a covalently closed circle Typically through back-splicing With no ends, these RNAs are much more stable as no exonuclease can act on them (potentially lasting days) They will still eventually degrade by endonuceolytic mechanisms like DNA- pairing & RNAse H circRNAs typically are not translatable While this could be simply accidental splicing, some genes produce circRNAs often enough to suggest functional relevance miRNA Sponges: circRNAs As many detected circRNAs can have multiple miRNA binding sites, they may be important in acting as competitive inhibitors for miRNAs, regulating how many can target mRNAs (miRNA sponges) Expression of a single siRNA can lead to degradation of the circRNA, rapidly releasing a payload of miRNAs Other non-circular RNAs called long non-coding (lncRNAs) may also act as miRNA sponges, but do not persist as long in the cell Summary miRNA and siRNA is produced in cells from a dsRNA intermediate siRNA typically from a viral infection miRNA from it’s own pri-miRNA gene or a mirtron Pri-miRNA converts to pre-miRNA by microprocessor complex & is exported Pre-miRNA is matured by Dicer miRNA is loaded into RISC to target mRNA Degradation if full-match Silencing if partial-match miRNAs can be regulated after production by circRNA/lncRNA Integrating Control RNAs into Gene Expression Integrating Control RNAs into Gene Expression And every gene product in this sequence could potentially be regulated by the same regulators listed here!  Copyright Sourcing Figures + Tables throughout PowerPoints from textbook. Permission: Courtesy of Freeman and Co./Macmillian Publishing Slide 3: Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990 Apr;2(4):279-289. doi: 10.1105/tpc.2.4.279. PMID: 12354959; PMCID: PMC159885. Slide 4: Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000. Early Development of the Nematode Caenorhabditis elegans. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10011/ Slide 5: Nobel Prize for Physiology or Medicine 2006. https://www.nobelprize.org/prizes/medicine/2006/popular-information/ Slide 6A: Abdallah, Florence and Chantal Pichon. “MicroRNAs in Skin Biology: Biogenesis, Regulations and Functions in Homeostasis and Diseases.” Immunome Research (2019): Slide 6B: Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G. 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Slide 25: Kyle Anderson for BMSC 320