Introduction to microRNA miRNA Workshop Ravi K. Gutti Division of Newborn Medicine Children´s Hospital Boston and Harvard Medical School Ravi. Gutti@childrens.harvard.edu May 18, 2010 http://catalyst.harvard.edu

Agenda Introduction and History Workflow in miRNA experiments Finding miRNA targets by target search databases miRNA validation and experimental considerations Applications

Why study miRNA?

Non-Coding RNA: Formerly known as “JUNK” 3

Non-Coding RNA: A Key to Eukaryotic Complexity? 4

What is miRNA?

Characteristics of miRNAs Small non-coding double stranded RNAs Approximately 19-22 nt long Repress activity of complementary mRNAs Regulate 30% of mammalian gene products 1 miRNA = hundreds of mRNAs “you can change a phenotype by modulating a single miRNA” Thomas Wurdinger, HMS Have been described in invertebrates and vertebrates: worms, fungi, plants, and mammals Many are conserved between vertebrates and invertebrates

History 7

History lin-4, first miRNA to be described in C. elegans; important in development of the worm from larva to adult. let-7, was also described in C. elegans (Reinhard BJ et al, 2000) as critical to stop the stem-cell-like divisions of seam cells and induce their fully differentiated state. Reduced let-7 expression is associated with human cancers and cancer stem cells, thus suggesting that let-7 in humans also promotes terminal differentiation and is a tumor suppressor. 1998-Fire and Mello, experiments in C. elegans, first to show that dsRNA is much more potent at inhibiting gene expression than antisense RNA. Set the stage for understanding the role of miRNAs in development and gene regulation. (Nobel Prize in Physiology and Medicine, 2007).

Genomic Organization 9

Biogenesis -Primary-miRNA is transcribed in the nucleus, and is usually several kilobases long; posses 5’ cap and a poly-A tail. -Cleaved in the nucleus by Drocha enzyme to 70nt hairpin transcript (pre-miRNA). -Transported to the cytoplasm by Exportin 5 through nuclear pores. -Cleaved by Dicer enzyme (RNase III enzyme) into 19-22nt ds-transcripts. -700 Human genes, 490 mouse genes. 1400 possible human sequences but 733 cloned or detected. Only ~120 tested. Esquela-Kerscher, A., and Slack, F.J. (2006). Nature Reviews 6, p. 263 10

siRNA miRNA

Why analyze miRNA patterns?

Why Analyze miRNA Expression Patterns? Complete role played by each sequence not yet determined • Functional analysis tools not yet widely available Knockdown, Over-Expression, Reporter Assays • Correlate miRNA expression profiles with: Biological Phenotypes, Protein & Gene Expression Levels • Discover miRNA sequences regulating genes involved in the biological process of interest

Relevance of miRNA to Human Biology Cancer. miRNAs have been found to be downregulated in a number of tumors , and in some cases the reintroduction of these miRNAs has been shown to impair the viability of cancer cells. The value of miRNA profiles in tumor diagnostics is well established. For instance, strong up and down regulations of 16 miRNAs have been shown in primary breast tumors, and these markers may aid in the development of drug-resistance and treatment-selection tests. Underlining the important role miRNA plays in oncology is the formation of several new companies which seek to expand development of miRNA-based therapeutics. Age-Related Diseases. Evidence is accumulating that many age-related diseases are associated with a decreased control of cell signaling that occurs in mid-life. The miRNA control of such systems as the cell cycle, DNA repair, oxidative stress responses and apoptosis, has been shown to become abnormally expressed in mid-life. It is highly likely that continued research will reveal important associations with the aging process, and may lead to therapeutics that can improve the quality of life. Heart Disease. Two heart-specific miRNAs were deleted in mouse models resulting in abnormal heart development in a large proportion of the offspring. While these lethal effects were expected, other studies show a more subtle role for miRNA in the heart. When miR-208 was eliminated, the mice appeared normal. Defects were revealed only when their hearts were stressed. These results show that comprehensive miRNA studies may be valuable in the diagnosis of heart disease. Neurological Diseases. Numerous reports have demonstrated the role of miRNAs in neural development. Evidence for a role in Parkinsons disease comes from animal model studies published last year, showing that loss of miRNAs may be involved in the development and progession of the disease. In cell culture experiments, transfer of small RNA fragments partially preserved miRNA deficient nerve cells. While these results and others point to an important role for miRNA in neurodegenerative disorders, much more work is needed to delineate the exact role of miRNAs in this important area. Immune Function Disorders. Recent miRNA deletion studies have revealed a central role in the regulation of the immune response. The deletion of miRNA-155 impaired T and B cell differentiation in germinal centers, and greatly decreased antibody and cytokine production. Two additional studies deleting miRNA-181 and 223 were found to control T cell response and granulocyte production, respectively. As more roles for miRNAs in the immune response are found, the list of immune function disorders with a miRNA component is certain to expand also.

miRNA Profiling Applications

Work flow in miRNA Experiments

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RNA Quality Assessment

Problems How do we find microRNA genes? Given a microRNA gene, how do we find its targets?

How do we find microRNA genes? Biological approach Small-RNA-cloning to identify new small RNAs Most microRNA genes are tissue-specific miR-124a is restricted to the brain and spinal cord in fish and mouse or to the ventral nerve cord in fly miR-1 is restricted to the muscles and the heart in mouse

miRNA Targets In plants, the identification of mRNA targets is straight forward because most miRNAs and their target mRNAs have exact or nearly exact complementarity. In animals, the tendency of miRNAs to bind their mRNA targets with imperfect sequence homology poses considerable challenges with target prediction. Several computational approaches have been developed to facilitate experimental design and predicting miRNA targets. Computational target prediction identifies potential binding sites according to base-pairing rules and across species conservation conditions.

MicroRNA Targets Prediction and Analysis

Target Identification The duplex for miRNA hsa-miR-579 and its target LRIG3 is partitioned into two parts, the seed part and the out-seed part Six to eight nucleotides at the 5’ end of the mature miRNA sequence are very important in the selection of target site Most of the computational tools developed to identify mRNA target sequences depend heavily on complementarity between miRNA seed sequence and the target sequence Most methods mainly use sequence complementarities, thermodynamic stability calculations and evolutionary conservation among species to determine the likelihood of formation of a productive miRNA-mRNA duplex

How to make more accurate predictions? Incorporating mRNA UTR structure to predict microRNA targets (Robins et al. 2005) Make sure the predicted target is “accessible”. Not forming basing pairing itself.

Other properties of microRNA targets MicroRNA targets are conserved across species. (Stark et al. 2003) Tend to appear in clusters. For lins, comparison is between C. elegans and C. briggsae. For hid, comparison is between D. melanogaster and D. pseudoobscura.

Another property of microRNA targets Sequence conservations of target sites Better complementary to the 5’ ends of the miRNAs.

Interesting properties of microRNA targets Clusters of microRNA targets Extensive co-occurrence of the sites for different microRNAs in target 3’ UTRs.

MicroRNA Targets Prediction Databases

Scientific Use Case Scenarios What are the target genes of a given miRNA? What are the miRNAs targeting a given gene and what are their binding sites? What is the tissue expression profile of a given miRNA (or set of miRNAs)? What is the miRNA expression profile in a given tissue (or set of tissues)?

Nat Methods. 2006 Nov;3(11):881-6 miRNA Tools

FEBS Journal 276 (2009) 2150–2156 miRNA Tools

Target prediction tools covered in today’s workshop Targetscan miRBase Argonaute

TargetScan (Lewis et al., Cell 2003) TargetScan predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA. As an option, nonconserved sites are also predicted. Also identified are sites with mismatches in the seed region that are compensated by conserved 3' pairing. In mammals, predictions are ranked based on the predicted efficacy of targeting as calculated using the context scores of the sites. TargetScanHuman considers matches to annotated human UTRs and their orthologs, as defined by UCSC whole-genome alignments. http://www.targetscan.org/

Targetscan (Lewis et al. Cell 2003) Given a microRNA that is conserved in multiple species and a set of orthologous 3’ UTR sequences: Use 7 nt segment of the miRNA as the ‘microRNA seed’ to find the perfect complementary motifs in the UTR regions. Extend each seeds to find the best energy Assign a Z score. Give a rank (Ri) according to that species. Repeat above process. Keep those genes for which Zi > Z_c and Ri < R_c.

miRBase Aims to provide integrated interfaces to comprehensive miRNA sequence data, annotation and predicted gene targets. miRBase takes over functionality from the miRNA Registry and fulfils three main roles: miRBase Sequences, miRBase Targets and miRBase Registry. http://microrna.sanger.ac.uk/

Sections of miRBase miRBase contains 3 main sections: miRBase Sequences contains all published miRNA sequences, genomic locations and associated annotation. miRBase Targets is a newly developed database of predicted miRNA target genes. miRBase Registry provides a confidential service assigning official names for novel miRNA genes prior to publication of their discovery

Argonaute (miRWalk) Nucleic Acids Res Argonaute (miRWalk) Nucleic Acids Res. 2006 Jan 1;34(Database issue):D115-8 Curated database Argonaute collects latest information from both literature and other databases. In contrast to databases on miRNAs like miRBase::Sequences, NONCODE or RNAdb, Argonaute hosts additional information on the origin of an miRNA, i.e. in which host gene it is encoded, its expression in different tissues and its known or proposed function, its potential target genes including Gene Ontology annotation, as well as miRNA families and proteins known to be involved in miRNA processing. Additionally, target genes are linked to an information retrieval system that provides comprehensive information from sequence databases and a simultaneous search of MEDLINE with all synonyms of a given gene. The web interface allows the user to get information for a single or multiple miRNAs, either selected or uploaded through a text file. Argonaute currently has information on miRNAs from human, mouse and rat. http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/interface

http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/interface

Single microRNA Module e.g. hsa-let-7a-1, mmu-mir-16-2, rno-mir-30c-2 HUMAN MOUSE RAT

Result for Single miRNA Exact Search

Origin Detail for Single miRNA Link to PubMed

Link to PubMed for each Reference

miRNA Target Validation and Considerations in RNAi Experiments

miRNA Target Validation Several in vitro methods useful to validate miRNA function: RNA interference protein analysis using SILAC Over-expression and knockdown functional studies  - There are three basic approaches to induce RNA interference: synthetic RNAi and siRNA duplexes vectors carrying RNAi cassette expressing shRNA or artificial miRNAs in vitro transcription and dicing of dsRNA to generate pools of siRNA. SILAC studies - Although miRNA profiling can show changes based on miRNA activity, it is not always indicative of translation inhibition. Protein analysis by mass spectrometry combined with stable isotopic labeling by amino acids in cell culture (SILAC) has strong correlation with miRNA activity. Relies upon the metabolic incorporation of 'light' or 'heavy' forms of the amino acid into the proteins to distinguish differences between cell populations (i.e. normal and malignant breast tissue)

Considerations for RNAi Experiments Method of siRNA production. If a good target sequence is unknown, or if you have had difficulty knocking down your gene using an oligonucleotide approach, use effective method such as transfecting a diced pool of siRNA and validate data using Stealth™ RNAi , siRNA or RNAi vectors. Delivery method(s)/cell type to use. Options for delivering siRNA include transfection and viral delivery. It is very important that the transfection efficiency be optimized. Even if an siRNA were100% effective (and most are only 70-95% effective), if only 50% of the cells are transfected you will never see better than a 50% knockdown. Select appropriate controls.

Considerations for RNAi Experiments Measure loss of message or protein function. The best method to verify loss of message is real time RT-PCR (much more sensitive than RT-PCR). If protein has long half-life, assays may need to be done at 48 or even 72 hours. Functionality. A visual assay can be done to identify a phenotypic change , such as looking for apoptotic cells. Alternatively, a functional cell based assay can be completed to analyze the effects of gene knockdown , such as a kinase or ion channel assay - or design your own cell based assay. Level of knockdown desired. If you are using a target that is knockdown to 70% or 80%, there may still be other potential targets that could result in more effective knockdown.

miRNA Applications

Examples of miRNA Functions & Relevance of miRNA to Human Biology

Role of miRNA in Disease and Cancer

Role of miRNA in Development and Neurodegeneration

miRNA in Cancer

Significance of miRNA

Potential miRNA Associated Products in Clinical Trials

Thank You

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