Mirco Castoldi, PhD
Post-transcriptional regulation of gene expression
Characterization of microRNA dysfunction in pathologic state
Since joining the group via a collaboration between the Children Hospital of the University of Heidelberg and the MMPU (Molecular Medicine Partnership Unit) I have been involved in the study of regulatory non-coding RNA. The central dogma of molecular biology defines how the flow of information moves from storage (DNA) to effectors (proteins). It states that DNA makes RNA and RNA makes protein and that the flow of information cannot move backwards (e.g. from protein to RNA or from RNA to DNA). Since it was stated this dogma has being challenged by several discoveries such as alternative splicing, retroviruses and prions. During the last decade the “dogma” has come once more under scrutiny by the discovery of microRNA (miRNA) and other non-coding RNA. Recently, it was shown that more than hundred thousand transcriptional units can be detected across the genome and that transcription not only occurs from those regions that encode for proteins (according the latest count the human genome contains approximately 20-23000 protein coding genes) but also within regions of the genome (e.g. intronic or intergenic regions) that in the past have been commonly labeled as “junk DNA”. A proportion of these transcriptional units codes for RNA transcripts that regulate conventional protein coding mRNAs. miRNAs are included within this category.
miRNAs constitute a class of short regulatory RNAs that control gene expression post-transcriptionally. miRNAs are initially transcribed as long precursor RNA molecules (pri-miRNA) that are successively processed by large RNA protein complexes termed Drosha and Dicer into their mature forms of ~ 22 nts (note that a proportion of miRNAs localized within introns of protein coding genes the pri-miRNAs is not processed by Drosha but by the spliceosome machinery and therefore these miRNAs are termed miRtrons). Cloning efforts and bioinformatics predictions suggest that miRNAs may regulate up to 30% of mammalian genes. miRNAs control mRNA expression at the level of turnover and/or translation via base pairing, usually to the 3'-untranslated regions. Hundreds of distinct miRNA genes are now known to exist and while for a large number of miRNAs the expression is equally detected across tissues for other the expression is spatially or temporally constricted to specific developmental stages or within tissue types (as example see Figure 1).
Overall, little is known about the biological function of animal miRNAs, but recent studies suggest important regulatory roles in a broad range of biological processes including developmental timing, cellular differentiation, proliferation and apoptosis. In most cases, however, the specific miRNA target genes are not known. Both, the qualitative and quantitative expression of miRNA is expected to regulate the transcriptome of a given cell or tissue. Therefore the accurate profiling of miRNA expression represents an important tool to investigate physiological and pathophysiological states.
Up to date more then 700 human miRNAs have been registered in miRbase (v10, October 2007) and their number is still on the rise, therefore to tackle the issue relate to the expression profiling of large number of genes we have developed a microarray platform for the expression profiling of mature miRNA named miChip. miChip technology accurately and sensitively monitors expression of miRNAs without prior need for RNA fractionation or miRNA amplification, and can discriminate between closely related miRNA family members. Our technology is based on the use of modified nucleotides, termed locked nucleic acid (LNA) that are incorporated within the oligonucleotide capture probes immobilized on the array surface. LNA is a synthetic RNA analog characterized by increased thermostability of nucleic acid duplexes when LNA monomers are introduced into oligonucleotides.
In the context of the MMPU we are exploiting miChip to investigate how diseased states affect miRNAs expression profiling. Our aim is to discover correlations between the changes in expression profiling (miRNA and mRNA) and disease prognosis. Currently we are involved in project addressing role of miRNA dysfunctions in childhood leukemia (TALL), brain cancer, breast cancer, prostate cancer, mesenchymal stem cells , heart diseases and iron metabolism.
To submit collaboration proposals for miRNA profiling please send a letter to: Mirco.Castoldi@embl.de
List of Related Publications
Mirco Castoldi, Sabine Schmidt, Vladimir Benes, Matthias W. Hentze, and Martina U. Muckenthaler. MiChip: An array-based method for microRNA expression profiling using locked nucleic acid (LNA) capture probes. Nature Protocols In Press.
Vladimir Benes, Jens Stolte, David Ibberson, Mirco Castoldi and Martina U. Muckenthaler (2007) Analysis of microRNA expression by qPCR. European Pharmaceutical Review 6, 27-29.
Mirco Castoldi, Vladimir Benes, Matthias W. Hentze and Martina U. Muckenthaler (2007) MiChip: a microarray platform for expression profiling of microRNAs based on locked-nucleic acid (LNA) oligonucleotide capture probe, Methods. 43, 146-152.
L. Venturini, K. Battmer, M. Castoldi, B. Schultheis, A. Hochhaus, M.U. Muckenthaler, A. Ganser, M. Eder, M. Scherr. (2007) Expression of the miR-17-92 polycistron in chronic myeloid leukemia (CML) CD34+ cells. Blood May 15;109(10):4399-4405.
Mirco Castoldi, Sabine Schmidt, Vladimir Benes, Mikkel Noerholm, Andreas E. Kulozik, Matthias W. Hentze, and Martina U. Muckenthaler (2006) A sensitive array for microRNA expression profiling (miChip) based on Locked Nucleic Acids (LNA). RNA. 12(5): 913-20.