Lesson Flowchart. Why Gene Expression Regulation?; microrna in Gene Regulation: an overview; microrna Genomics and Biogenesis;

micrornas

Lesson Flowchart Why Gene Expression Regulation?; microrna in Gene Regulation: an overview; microrna Genomics and Biogenesis; How do micrornas function? micrornas and complex cellular circuits; micrornas and TFs; micrornas and pathology; micrornas as potential therapeuitics; How to study micrornas In silico and In vitro

Why Gene Expression Regulation?

RECONTRUCTING THE FUNCTIONING HUMAN GENOME A complete description of the DNA sequence of an organism -be it the few million nucleotides of a bacterium or the few billion nucleotides of a human -would no more enable us to reconstruct the organism than a list of English words would enable us to reconstruct a play by Shakespeare. In both cases the problem is to know how the elements in the DNA sequence or the words on the list are used. Under what conditions is each gene product made, and, once made, what does it do? Alberts B et al. Mol Biol of the Cell 4th ed

AS IN CIVIL SOCIETY, WHERE THERE MUST necessarily be checks and balances on freedom of expression, cells have evolved a range of mechanisms to regulate the expression of their constituent genes. GUY RIDDIHOUGH, BEVERLY A. PURNELL, JOHN TRAVIS SCIENCE VOL 319 28 MARCH 2008

GENE EXPRESSION FLOWCHART mirna Nuclear phase Choise and Transcription of specific genes RNA Maturation Nucleus-Cytoplasm transfer Cytoplasmatic phase Protein Synthesis Post-translational modifications Protein travelling

The great group of non-coding RNA NON-CODING RNA SMALL RNA LONG nc RNA microrna pirna snrna rrna snorna trna Other ncrna

The overwhelming regulatory potential of RNA

microrna are as important in physiology as well as in pathology

microrna in Gene Regulation: an overview

WHAT IS A microrna? MicroRNAs (mirnas) are endogenous ~22 nt long RNAs that can play important regulatory roles in animals and plants by targeting mrnas for cleavage or translational repression. By this way they can influence the output of many protein-coding genes. [David P. Bartel, Cell, Vol. 116, 281 297, January 23, 2004]

mirnas FEATURES They have quite all evolutionary conserved sequences, suggesting that these molecules participate in essential processes; Often they have a space and/or temporal specific patterns of expression profiles; They could be localized either in intergenic region or in genic region (usually in introns or in non-coding exons); They could be organized into cluster genes

mirnas FEATURES They specify hetero-silencing, in that they are produced from genes that specify the silencing of very different genes; The same mirna could target different types of mrnas; Different types of mirnas could target the same mrna; In H. sapiens they are predicted to target over one third of all genes

SO micrornas CONSTITUTE A NEW LEVEL OF REGULATION AND FINE-TUNING OF GENE EXPRESSION

MAIN FUNCTIONS OF mirnas Cell proliferation; Cell death; Fat Metabolism In Flies (Brennecke et al., 2003; Xu et al., 2003) Neuronal patterning In Nematodes (Johnston and Hobert, 2003) Modulation of Hematopoietic Lineage differentiation In Mammals (Chen et al., 2004) Control of Leaf and Flower Development In plants (Palatnik et al., 2003)

To comprehend the importance of mirnas we can argue the experiment performed By Dr. Deepak Srivastava (Director of the Gladstone Institute of Cardiovascular Diseases University of California, San Francisco). She engineered mice lacking mir-1-2, normally expressed in heart at high levels. No great differences between engineered and normal mice was expected because mir-1-2 has a twin, an identical DNA sequence on another chromosome that the Researchers didn t delete. By knocking out one copy, they dialed down the dose of this microrna by 50%. This was sufficient to dye half the animals for either holes in the heart or fatal disruptions in cardiac rhythm (Science, 27 April 2007, p.530). By these experiments scientists are now learning that micrornas are the regulators of the master regulators, potentially the controllers of entire pathways of genes.

mirnas TIMELINE

irna TIMELINE

A BIT OF STATISTICS Each mirna may potentially bind to as many as 200 targets, and it s estimated that mirnas control the expression of at least one third of human mrnas. http://mirnamap.mbc.nctu.edu.tw/

mirbase 706 mirnas in H. sapiens is last update, even if more than 1000 are computationally predicted

A GROWING BULK OF DATA

microrna Genomics and Biogenesis

GENOMICS OF micrornas Zhao Y and Srivastava D, 2007 (In press)

mirna GENOMICS: THE CLUSTER ORGANIZATION Ex.: mir-15a-mir-16 Cluster falls within a region of human chr. 13 (13q14), thought to harbor a tumor suppressor gene, because it s the site of the most common structural aberrations in both mantle cell lymphoma and B cell chronic lymphocytic Leukemia. Mariana Lagos-Quintana, et al. (2001)

microrna BIOGENESIS: IT S A LONG WAY!

DROSHA: CROPPING OF PRI-microRNAs DROSHA IS A NUCLEAR RNase III; IT FORMS A LARGE COMPLEX OF ~ 650 KD IN HUMANS WITH THE DI GEORGE SYNDROME CRITICAL REGION GENE 8 (DGCRB) PROTEIN

microrna BIOGENESIS: IT S A LONG WAY!

DICER: DICING OF PRE-microRNAs DICER IS A CYTOPLASMIC RNase III; N-TERMINAL SEGMENT CONTAINS A PAZ DOMAIN (THE SAME FOUND IN ARGONAUTE PROTEINS), RESPONSABLE FOR THE BINDING OF THE 3 OVERHANG OF THE PRE-microRNAs

microrna BIOGENESIS: IT S A LONG WAY!

BIOGENESIS OF mirna: TRANSCRIPTION A. Plants B. Metazoa RNase III Endonuclease C. Animal sirnas RNase III Endonuclease Bartel D P, 2004

STRAND SELECTION OF MATURE microrna MATURE MIRNAS ARE INCORPORATED INTO EFFECTOR COMPLEXES THAT ARE KNOWN AS MIRNP (MIRNA- CONTAINING RIBONUCLEOPROTEIN COMPLEX), MIRGONAUTE OR MIRISC (MIRNA-CONTAINING USUALLY ONLY THE STRAND OF THE RNA-INDUCED SILENCING DUPLEX WITH REALTIVELY UNSTABLE COMPLEX). BASE PAIRS AT THE 5 END TYPICALLY REMAINS!!!

STRAND SELECTION: THERMODYNAMICS RULES! 5 GGUGCUACGGUUGCCAUAAAU 3 3 CCCCACGAUGCCAACGGUAUU 5 3 SEED ACGGUAU 5

How do micrornas function?

Canonical Binding Between micrornas and 3 UTR of the Gene Target

RNAi PATHWAY: THE COMMON WAY OF mirnas AND sirnas NOBEL PRIZE

mir-148 targets human DNMT3b protein coding region mir-148 targets Dnmt3b splice variants 1,2 and 4 but not 3, because the latter doen t contain the exon containing its binding site Duursma A M et al., RNA, 2008

MECHANISMS OF microrna ACTION 2 POSSIBLE MECHANISMS: 1) TRANSLATIONAL REPRESSION; 2) mrna CLEAVAGE

micrornas and complex cellular circuits

FAVOURITE mirna TARGETS Ligands Cell-surface receptors Intracellular central signaling proteins Fraction of mirna target 9.1% (3/33) 31.2% (122/391) 18.8% (15/80) Nuclear proteins 50.0% (19/38) mirnas predominantly target positive regulatory motifs, highly connected scaffolds and most downstream network components such as signaling transcription factors, but less frequently target negative regulatory motifs, common components of basic cellular machines and most upstream network components such as ligands. Cui Q. er al, 2006 modified

Are mirnas only repressive elements?...it depends on the Circuit! DIFFERENTIATED CELL UNDIFFERENTIATED CELL Tsang J et al., Mol Cell 26,753-767, June 2007

C Tsang J et al., Mol Cell 26,753-767, June 2007

Multilevel Regulation of Gene Expression by MicroRNAs Recent studies indicate that tissue specific mirnas may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mrna splicing. This multilevel regulation may allow individual mirnas to profoundly affect the gene expression program of differentiated cells. Eugene V. Makeyev and Tom Maniatis, Science 2008

A number of mirna targets fall into the category of gene batteries, sets of functionally related effector genes that represent outputs of gene regulatory networks mir-124 is neuronspecific; REST is a suppressor of neuron-specific genes See Eugene V. Makeyev, et al. Science 319, 1789 (2008)

micrornas can also influence splicing PTBP1 and 2 are repressors of alternative splicing (even if PTBT1 is more repressive than 2) that in early neural cell precursors repress the inclusion of a number of neuron specific exons into mature mrnas. PTBP1 functions in this context as a repressor of neuron-specific alternative exon inclusion, and it is expressed in neural precursors as well as many other types of nonneuronal cells. In differentiating and mature neurons, PTBP1 quantities decrease, leading to the inclusion of a number of neuron-specific alternative exons in mature mrna. Eugene V. Makeyev, et al. Science 319, 1789 (2008)

The increasing diversity of cellular differentiation in metazoans, accompanied by an increase in the complexity of gene regulatory networks, must have required a mechanism to prevent interference between spatially or temporally adjacent gene expression programs. The above examples argue that at least some mirnas play an important role in this mechanism by effectively rewiring the cell-specific networks at all levels of the regulatory hierarchy, from the gene battery to global regulators of transcription and alternative splicing.

The analysis extended to five vertebrates, a fly and a nematode. shows that the mirna target density for neuronal transcripts linearly increases along the evolutionary scale, reaching a maximum in mammals. On the contrary, the mirna target site density in the 3'UTRs of r-protein transcripts remained similar in all animals. These findings indicate that transcripts specifically expressed during neurogenesis had a selective evolutionary advantage in acquiring mirna target sites, supporting the notion that mirna-dependent regulatory circuitries act as molecular driving forces for increasing functional complexity

micrornas and TFs

Pleiotropic, Combinatorial, Cooperative, Accessibility, Regulative and Network Motifs effects of mirnas and TFs

But are mirna and TFs really on par in terms of their importance for gene regulatory events? The deletion of >80% of individual mirna loci in Caenorhabiditis elegans revealed that less than 10% of mirna knockouts result in clear developmental or morphological defects. In contrast, RNA interference mediated loss-offunction analysis shows that about 30% of all C. elegans TF losses cause easily observable phenotypes. See E. A. Miska et al., PLoS Genet 3, e215 (2007) and www.wormbase.org

Are there intrinsic features of mirnas that would explain why they may cover a more restricted regulatory niche as compared to TFs? The gene regulatory region of TFtargets is oftencomplex and can span dozens of kilobases, whereas mirna-controlled 3 - untranslated regions are, on average, <1 kb in size. Not only does this limit the amount of regulatory inputs that a gene can sample from mirnas versus TFs, but it also provides less substrate for evolution to play on, i.e., to evolve new regulatory inputs, a major driving force in evolution. SeeO.Hobertetal,Science319,1785(2008)

A NOTE ON FUNCTION Working Model for the Roles of mirnas that Target the Messages of Transcription Factors during Plant Development. Following cell division, the daughter cells inherit mrnas from the precursor cell (step 1). A differentiating daughter cell (cell on right) expresses new transcription factor messages (green) as well as a mirna (red) complementary to messages that must be cleared (blue) in order for the cell to progress to the differentiated state (step 2). The mirna directs the cleavage of target messages, preventing prolonged or inappropriate expression of the transcriptional regulator, thus enabling the rapid differentiation of the daughter cell (step 3). (Figure redrawn from Rhoades et al., 2002, copyrighted by Cell press, used with permission.)

micrornas and pathology

Human microrna genes are frequently located at fragile sites and genomic regions involved in cancers Calin G A et al., PNAS March 2, 2004 vol. 101 no. 9 2999 3004

micrornas CAN FUNCTION AS TS AND OG

Possible effects of altering microrna expression Science, 28 March 2008, Vol. 319 MicroRNAs Make Big Impression In Disease After Disease

Glc mir-30d Insulin Glc mir-30d Insulin

A recent study (Cogswell J P et al., J Alz Dis, 2008) demonstrated the importance of deregulation of mirnas expression in brains and cerebrospinal fluid (CSF) of Alzheimer s patients and proposed to use these modifications as putative biomarkers of the disease

The commercial face of microrna discovery Science, 28 March 2008, Vol. 319 MicroRNAs Make Big Impression In Disease After Disease

micrornas as potential therapeuitics

Possible microrna therapy Stoffel studies micrornas in diabetes and metabolism and realized that he needed a delivery system to get RNA sequences into cells where they could silence micrornas.to accomplish this, Stoffel collaborated with Muthiah Manoharan of Alnylam Pharmaceuticals, an RNAi company in Cambridge, Massachusetts, to create antagomirs. Stoffel s antagomirs are RNA snippets linked to cholesterol molecules, which help slip the silencers into cells. After being injected into the tail veins of mice, antagomirs travel through the body; they have successfully modified microrna expression in many organs. Antagomirs can t cross the blood-brain barrier, but scientists have injected them directly into the brain, where they penetrated brain cells. By the third day after an antagomir injection, Stoffel says, the micrornas targeted disappear and stay silent for weeks.

The cardiology field has an advantage over others: Its doctors have experience supplying drugs straight into the target organ,for example, by injection into the coronary arteries. It should be doable, some predict, to shoot extra micrornas, or microrna suppressors, directly into the heart. I think we might see the first trials [of a microrna-based therapy] in the cardiology field, says Markus Stoffel, a molecular biologist at the Swiss Federal Institute of Technology in Zürich. Science, 28 March 2008, Vol. 319 MicroRNAs Make Big Impression In Disease After Disease

How to study micrornas In silico and In vitro

micrornas EXPRESSION STUDIES: A FIRST STEP TO UNDERSTAND THE FUNCTION THESE ARE TECHNIQUES USED FOR IN VITRO ANALYSES OF micrornas GENE EXPRESSION!

HOCTAR: Host gene Oppositely Correlated TARgets Gennarino V A et al., Genome Res, December 2008

TaqMan MicroRNA Assays The TaqMan MicroRNA Assays are designed to detect and accurately quantify mature micrornas (mirnas)

TaqMan MicroRNA Assays offer several distinct advantages over conventional mirna-detection methods, including: High-quality quantitative data The assays can detect and quantify mirna over more than six logs of dynamic range. Sensitivity The assays can detect mirnas in as little as 1 to 10 ng of total RNA, allowing you to conserve limited samples. High Specificity The assays detect only mature mirna, not its precursor, with single-base discrimination. Fast and simple methodology The two-step protocol takes less than four hours and can be used with any Applied Biosystems Real-Time PCR instrument.

The TaqMan MicroRNA Assays use looped-primer RT-PCR, a new real-time quantification method, to accurately detect mature mirnas. Each TaqMan MicroRNA assay includes: One tube containing mirna-specific RT primer One tube containing a mix of: mirna-specific forward PCR primer specific reverse PCR primer mirna-specific TaqMan MGB probe