microRNAmicroRNAmicroRNAmicroRNA

Cass JabaraCass JabaraCass JabaraCass Jabara

Exam QuestionsExam QuestionsExam QuestionsExam Questions

What are microRNAs, and what is What are microRNAs, and what is their primary function?their primary function?

Where are microRNAs commonly Where are microRNAs commonly found in the human genome, and found in the human genome, and how does this correlate to disease?how does this correlate to disease?

What are microRNAs, and what is What are microRNAs, and what is their primary function?their primary function?

Where are microRNAs commonly Where are microRNAs commonly found in the human genome, and found in the human genome, and how does this correlate to disease?how does this correlate to disease?

BackgroundBackgroundBackgroundBackground

miRNA - miR - microRNAmiRNA - miR - microRNAmiRNA - miR - microRNAmiRNA - miR - microRNA

EukaryotesEukaryotes S.S. RNAS.S. RNA 19- to 25-nt19- to 25-nt Non-codingNon-coding

Regulates gene expressionRegulates gene expression

Evolutionary conservedEvolutionary conserved

EukaryotesEukaryotes S.S. RNAS.S. RNA 19- to 25-nt19- to 25-nt Non-codingNon-coding

Regulates gene expressionRegulates gene expression

Evolutionary conservedEvolutionary conserved

≠ ≠ mRNA!mRNA!

Eukaryotic GenomeEukaryotic GenomeEukaryotic GenomeEukaryotic Genome

1/3 of genes1/3 of genes regulatedregulated

222 human miRNAs

ProcessingProcessingProcessingProcessing

DNADNA

pri-miRNApri-miRNA

pre-miRNApre-miRNAExportin 5-induced exportExportin 5-induced export

NUCLEUSNUCLEUS CYTOPLASM CYTOPLASM

transcriptiontranscription

Drosha processingDrosha processingDicer processingDicer processing

maturematuremiRNAmiRNA

RISCRISC

miRNA FunctionmiRNA FunctionmiRNA FunctionmiRNA Function

3’3’ 5’5’AAAAAAAAAAAAAAAAAAAAAA5’5’ 3’3’ mRNAmRNA

TranslationTranslation

miRNA FunctionsmiRNA FunctionsmiRNA FunctionsmiRNA Functions

Cellular division - coordinationCellular division - coordination Developmental apoptosisDevelopmental apoptosis Stress resistanceStress resistance Fat metabolismFat metabolism

Positive or negative regulationPositive or negative regulation

Cellular division - coordinationCellular division - coordination Developmental apoptosisDevelopmental apoptosis Stress resistanceStress resistance Fat metabolismFat metabolism

Positive or negative regulationPositive or negative regulation

Primary LiteraturePrimary LiteraturePrimary LiteraturePrimary Literature

Novel miRNA IDNovel miRNA IDNovel miRNA IDNovel miRNA ID

BioinformaticsBioinformatics Scan for conserved hairpin structureScan for conserved hairpin structure

Conserved, repetitive, protein codingConserved, repetitive, protein codingThermodynamic stabilityThermodynamic stabilityExpression of predicted miRNAs - Expression of predicted miRNAs -

microarraysmicroarraysValidate microarray hits via cloning and Validate microarray hits via cloning and

sequencing, fish via capture biotinylated sequencing, fish via capture biotinylated oligonucleotidesoligonucleotides

Placenta, testis, thymus, brain, prostatePlacenta, testis, thymus, brain, prostate

BioinformaticsBioinformatics Scan for conserved hairpin structureScan for conserved hairpin structure

Conserved, repetitive, protein codingConserved, repetitive, protein codingThermodynamic stabilityThermodynamic stabilityExpression of predicted miRNAs - Expression of predicted miRNAs -

microarraysmicroarraysValidate microarray hits via cloning and Validate microarray hits via cloning and

sequencing, fish via capture biotinylated sequencing, fish via capture biotinylated oligonucleotidesoligonucleotides

Placenta, testis, thymus, brain, prostatePlacenta, testis, thymus, brain, prostate

Bentwich et. al, 2005

Oligonucleotide CaptureOligonucleotide CaptureOligonucleotide CaptureOligonucleotide Capture

miRNA ClustersmiRNA ClustersmiRNA ClustersmiRNA Clusters

Cluster 1 - Chromosome 19 - placenta onlyCluster 1 - Chromosome 19 - placenta only 54 miRNAs - largest cluster discovered54 miRNAs - largest cluster discovered Expression parallels human embryonic stem Expression parallels human embryonic stem

cellscells Conserved in primates onlyConserved in primates only

Cluster 2 - X Chromosome - testis onlyCluster 2 - X Chromosome - testis only 10 miRNAs10 miRNAs

10 conserved in primates only10 conserved in primates only 7 conserved in dog7 conserved in dog 4 conserved in mouse and rat4 conserved in mouse and rat

Cluster 1 - Chromosome 19 - placenta onlyCluster 1 - Chromosome 19 - placenta only 54 miRNAs - largest cluster discovered54 miRNAs - largest cluster discovered Expression parallels human embryonic stem Expression parallels human embryonic stem

cellscells Conserved in primates onlyConserved in primates only

Cluster 2 - X Chromosome - testis onlyCluster 2 - X Chromosome - testis only 10 miRNAs10 miRNAs

10 conserved in primates only10 conserved in primates only 7 conserved in dog7 conserved in dog 4 conserved in mouse and rat4 conserved in mouse and rat

SignificanceSignificanceSignificanceSignificance Human miRNA total >800Human miRNA total >800

(vs current knowledge of 222)(vs current knowledge of 222) Conserved only 400-500Conserved only 400-500

Significant miRNA evolution unique to Significant miRNA evolution unique to primatesprimates 89 Novel miRNAs discovered89 Novel miRNAs discovered

53 not conserved under primates53 not conserved under primates

Primate-specific developmental Primate-specific developmental expression indicates important role in expression indicates important role in mammalian complexitymammalian complexity

Human miRNA total >800Human miRNA total >800 (vs current knowledge of 222)(vs current knowledge of 222) Conserved only 400-500Conserved only 400-500

Significant miRNA evolution unique to Significant miRNA evolution unique to primatesprimates 89 Novel miRNAs discovered89 Novel miRNAs discovered

53 not conserved under primates53 not conserved under primates

Primate-specific developmental Primate-specific developmental expression indicates important role in expression indicates important role in mammalian complexitymammalian complexity

miRNA and CancermiRNA and CancermiRNA and CancermiRNA and Cancer

Novel tumorigenesis gene classNovel tumorigenesis gene classe.g. 13q14 deletions - e.g. 13q14 deletions - miR-15amiR-15a, , miR-miR-

16a16aB cell chronic lymphocytic leukemiaB cell chronic lymphocytic leukemiaMantle cell lymphomaMantle cell lymphomaMultiple myelomaMultiple myelomaProstate cancerProstate cancer

Novel tumorigenesis gene classNovel tumorigenesis gene classe.g. 13q14 deletions - e.g. 13q14 deletions - miR-15amiR-15a, , miR-miR-

16a16aB cell chronic lymphocytic leukemiaB cell chronic lymphocytic leukemiaMantle cell lymphomaMantle cell lymphomaMultiple myelomaMultiple myelomaProstate cancerProstate cancer

miRNA LocationsmiRNA LocationsmiRNA LocationsmiRNA Locations

HPV Integration SitesHPV Integration Sites

FRAs - Fragile SitesFRAs - Fragile Sites Loci prone to metaphase breakageLoci prone to metaphase breakage Rearrangement or deletions in cancerRearrangement or deletions in cancer

CAGR - Cancer-associated genomic regionsCAGR - Cancer-associated genomic regions LOH - loss of heterozygosity (e.g. p53, RB)LOH - loss of heterozygosity (e.g. p53, RB)

HOX - homeobox clustersHOX - homeobox clusters Developmental gene regulationDevelopmental gene regulation

HPV Integration SitesHPV Integration Sites

FRAs - Fragile SitesFRAs - Fragile Sites Loci prone to metaphase breakageLoci prone to metaphase breakage Rearrangement or deletions in cancerRearrangement or deletions in cancer

CAGR - Cancer-associated genomic regionsCAGR - Cancer-associated genomic regions LOH - loss of heterozygosity (e.g. p53, RB)LOH - loss of heterozygosity (e.g. p53, RB)

HOX - homeobox clustersHOX - homeobox clusters Developmental gene regulationDevelopmental gene regulation

oncogenes

Calin et. al, 2003

SummarySummarySummarySummary

microRNAs - 19-25nt - regulates microRNAs - 19-25nt - regulates gene expressiongene expression

Over 800 predicted, many primate Over 800 predicted, many primate conserved, may play role in conserved, may play role in complexitycomplexity

Positive and negative regulation, Positive and negative regulation, novel tumorigenesis gene classnovel tumorigenesis gene class

microRNAs - 19-25nt - regulates microRNAs - 19-25nt - regulates gene expressiongene expression

Over 800 predicted, many primate Over 800 predicted, many primate conserved, may play role in conserved, may play role in complexitycomplexity

Positive and negative regulation, Positive and negative regulation, novel tumorigenesis gene classnovel tumorigenesis gene class

ReferencesReferencesReferencesReferences Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O.,

Barzilai, A., Einat, P., Einav., E., Meiri., E., Sharon, E., Spector, Y., Barzilai, A., Einat, P., Einav., E., Meiri., E., Sharon, E., Spector, Y., Bentwich, Z. (2005) Identification of hundreds of conserved and Bentwich, Z. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. nonconserved human microRNAs. NatureNature 77: 766-770.: 766-770.

Calin, G.A., Sevignani, C, Dumitru, C.D., Hyslop, T., Noch, E., Yendamuri, 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) S., Shimizu, M., Rattan, S., Bullrich, H., Negrini, M., Croce, C.M. (2003) Human microRNA genes are frequently located at fragile sites and Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. genomic regions involved in cancers. Proc. Natl. Acad. Sci.Proc. Natl. Acad. Sci. 101101: 2999-: 2999-30043004

““microRNA (miRNA) Resource” Ambion. microRNA (miRNA) Resource” Ambion. http://www.ambion.com/techlib/resources/miRNA/index.htmlhttp://www.ambion.com/techlib/resources/miRNA/index.html

““MicroRNA” Wikipedia. MicroRNA” Wikipedia. http://en.wikipedia.org/wiki/MicroRNAhttp://en.wikipedia.org/wiki/MicroRNA

““RNA-induced Silencing Complex” Wikipedia. RNA-induced Silencing Complex” Wikipedia. http://en.wikipedia.org/wiki/RNA-induced_silencing_complexhttp://en.wikipedia.org/wiki/RNA-induced_silencing_complex

Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Barzilai, A., Einat, P., Einav., E., Meiri., E., Sharon, E., Spector, Y., Barzilai, A., Einat, P., Einav., E., Meiri., E., Sharon, E., Spector, Y., Bentwich, Z. (2005) Identification of hundreds of conserved and Bentwich, Z. (2005) Identification of hundreds of conserved and nonconserved human microRNAs. nonconserved human microRNAs. NatureNature 77: 766-770.: 766-770.

Calin, G.A., Sevignani, C, Dumitru, C.D., Hyslop, T., Noch, E., Yendamuri, 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) S., Shimizu, M., Rattan, S., Bullrich, H., Negrini, M., Croce, C.M. (2003) Human microRNA genes are frequently located at fragile sites and Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. genomic regions involved in cancers. Proc. Natl. Acad. Sci.Proc. Natl. Acad. Sci. 101101: 2999-: 2999-30043004

““microRNA (miRNA) Resource” Ambion. microRNA (miRNA) Resource” Ambion. http://www.ambion.com/techlib/resources/miRNA/index.htmlhttp://www.ambion.com/techlib/resources/miRNA/index.html

““MicroRNA” Wikipedia. MicroRNA” Wikipedia. http://en.wikipedia.org/wiki/MicroRNAhttp://en.wikipedia.org/wiki/MicroRNA

““RNA-induced Silencing Complex” Wikipedia. RNA-induced Silencing Complex” Wikipedia. http://en.wikipedia.org/wiki/RNA-induced_silencing_complexhttp://en.wikipedia.org/wiki/RNA-induced_silencing_complex