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microRNA

microRNA. Cass Jabara. Exam Questions. What are microRNAs, and what is their primary function? Where are microRNAs commonly found in the human genome, and how does this correlate to disease?. Background. miRNA - miR - microRNA. Eukaryotes S.S. RNA 19- to 25-nt Non-coding

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microRNA

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  1. microRNA Cass Jabara

  2. Exam Questions • What are microRNAs, and what is their primary function? • Where are microRNAs commonly found in the human genome, and how does this correlate to disease?

  3. Background

  4. miRNA - miR - microRNA • Eukaryotes • S.S. RNA • 19- to 25-nt • Non-coding • Regulates gene expression • Evolutionary conserved ≠ mRNA!

  5. 222 human miRNAs 1/3 of genes regulated Eukaryotic Genome

  6. Processing DNA RISC transcription pri-miRNA mature miRNA Drosha processing Dicer processing pre-miRNA Exportin 5-induced export NUCLEUS CYTOPLASM

  7. 5’ mRNA AAAAAAAAAAA 3’ 3’ 5’ miRNA Function Translation

  8. miRNA Functions • Cellular division - coordination • Developmental apoptosis • Stress resistance • Fat metabolism • Positive or negative regulation

  9. Primary Literature

  10. Novel miRNA ID • Bioinformatics • Scan for conserved hairpin structure • Conserved, repetitive, protein coding • Thermodynamic stability • Expression of predicted miRNAs - microarrays • Validate microarray hits via cloning and sequencing, fish via capture biotinylated oligonucleotides • Placenta, testis, thymus, brain, prostate

  11. Oligonucleotide Capture Bentwich et. al, 2005

  12. miRNA Clusters • Cluster 1 - Chromosome 19 - placenta only • 54 miRNAs - largest cluster discovered • Expression parallels human embryonic stem cells • Conserved in primates only • Cluster 2 - X Chromosome - testis only • 10 miRNAs • 10 conserved in primates only • 7 conserved in dog • 4 conserved in mouse and rat

  13. Significance • Human miRNA total >800 • (vs current knowledge of 222) • Conserved only 400-500 • Significant miRNA evolution unique to primates • 89 Novel miRNAs discovered • 53 not conserved under primates • Primate-specific developmental expression indicates important role in mammalian complexity

  14. miRNA and Cancer • Novel tumorigenesis gene class • e.g. 13q14 deletions - miR-15a, miR-16a • B cell chronic lymphocytic leukemia • Mantle cell lymphoma • Multiple myeloma • Prostate cancer

  15. oncogenes miRNA Locations • HPV Integration Sites • FRAs - Fragile Sites • Loci prone to metaphase breakage • Rearrangement or deletions in cancer • CAGR - Cancer-associated genomic regions • LOH - loss of heterozygosity (e.g. p53, RB) • HOX - homeobox clusters • Developmental gene regulation

  16. Calin et. al, 2003

  17. Summary • microRNAs - 19-25nt - regulates gene expression • Over 800 predicted, many primate conserved, may play role in complexity • Positive and negative regulation, novel tumorigenesis gene class

  18. References • Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Barzilai, A., Einat, P., Einav., E., Meiri., E., Sharon, E., Spector, Y., Bentwich, Z. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nature7: 766-770. • Calin, G.A., Sevignani, C, Dumitru, C.D., Hyslop, T., Noch, E., Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, H., Negrini, M., Croce, C.M. (2003) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci.101: 2999-3004 • “microRNA (miRNA) Resource” Ambion. http://www.ambion.com/techlib/resources/miRNA/index.html • “MicroRNA” Wikipedia. http://en.wikipedia.org/wiki/MicroRNA • “RNA-induced Silencing Complex” Wikipedia. http://en.wikipedia.org/wiki/RNA-induced_silencing_complex

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