Visualizing miRNA Expression: Strategies and Applications
Tuesday, October 3, 2006
Presented by the RNAi Discussion Group
Organizers: Thomas Tuschl, The Rockefeller University and Eric Lai, Sloan-Kettering Institute
Speakers: Zissimos Mourelatos, University of Pennsylvania; Wigard Kloosterman, Netherlands Institute for Developmental Biology; Eric Lai, Memorial Sloan-Kettering Cancer Center; Pablo Landgraf, The Rockefeller University
A major component of the effort to understand miRNA function is to understand in detail the specific tissues, organs and cells that express individual miRNAs. This meeting will highlight different methods for determining miRNA expression patterns, and how such knowledge can be applied to elucidate miRNA function.
Tools and strategies to analyze microRNAs: Zissimos Mourelatos
MicroRNAs (miRNAs) are small regulatory RNAs that assemble with Argonaute proteins in RNA Induced Silencing Complexes (RISCs)/miRNPs and function by base pairing with mRNA targets. Strategies and technologies to profile and visualize miRNAs and their application to studying human tissues will be discussed. The properties of a new anti-Argonaute monoclonal antibody will be presented along with a UV-crosslinking method to study direct interactions between miRNPs and mRNA targets in human cells.
Cell-type and tissue-specific miRNAs determined by small RNA library cloning: Pablo Landgraf
MicroRNAs (miRNAs) are a recent discovered class of conserved 21-23 nucleotide short non-coding RNAs regulating protein levels by translational inhibition and mRNA destabilization and are involved in a variety of processes including cell differentiation and apoptosis. In order to accurately identify miRNAs and their specificity of expression, we prepared and analyzed over 250 small RNA libraries that originate from 26 different organ systems and cell types of human and rodents. The sample set is particularly rich for neuronal and normal and malignant hematopoietic cells and tissues. Computational tools were developed to analyze and present miRNA expression profiles produced by conventional sequencing of about 1,000 miRNA clones per library. The large total number of clones enabled a general analysis of miRNA processing patterns and lead to sequence adjustment of a significant number of previously reported miRNA sequences. In total we identified 333, 295 and 196 distinct mature miRNAs by cloning and sequencing from human, mouse and rat encoded by 389, 358 and 220 different miRNA genes, respectively. These miRNA genes are organized in 204, 183 and 136 transcription units, respectively, due to polycistronic organization of many miRNA genes. Combining information obtained from evolutionary conservation, cloning frequencies, processing patterns and genomic sequence analysis, we classified miRNAs to prioritize and guide future bioinformatic and experimental functional analysis. (The analysis included RNA samples provided by many collaborators that are not listed as authors in this abstract.)
Drosophila microRNAs: from genes to functions:Eric Lai
We are using the Drosophila system to understand the biological functions of microRNAs. We first combined computational and molecular approaches to catalog the microRNAs in this organism. We are now applying systematic tests to identify microRNAs that may influence developmental patterning. These tests include in situ hybridization to identify their spatial domains of expression, and gain-of-function studies to reveal their activities in vivo.