Research Report 2010 - MDC

PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation)Photoactivatable 4-Thiouridine (4SU), when providedto cultured cells, is incorporated into nascentRNAs and crosslinked to RNA-interacting proteins bylong-wave UV irradiation. The protein of interest isimmunoprecipitated and the covalently bound RNAreduced to a short protected segment by ribonucleasetreatment. In contrast to other methods, thesites of crosslinking are identified by mapping T to Ctransitions residing in the cDNA of libraries preparedfrom RNA crosslinked to a specific RNA-interactingprotein.Regulation of gene expression by non-codingRNAsNon-coding RNA (ncRNA) are functional RNAs that arenot translated into proteins. Recent evidence indicatesthat sizable regions of mammalian genomes are transcribedinto ncRNAs, which are alternatively splicedand/or processed into smaller RNAs. microRNAs, themost prominent class of small ncRNAs, mediate destabilizationand inhibition of translation target mRNAs.We are using next generation sequence technology todiscover large ncRNAs in human cells lines. To obtaininsights into the function of identified ncRNAs wedeplete these RNAs by RNA interference and characterizeassociated proteins by photo-crosslinking and massspectrometry.Selected PublicationsHausser J*, Landthaler M*, Jaskiewicz L, Gaidatzis D, Zavolan M. (2009)Relative contribution of sequence and structure features to the mRNAbindingof Argonaute/miRNA complexes and the degradation of miRNAtargets. Genome Res 19. 2009-2020.Zhang Y, Landthaler M, Schlussman SD, Yuferov V, Ho A, Tuschl T, KreekMJ.(2009) Mu opioid receptor knockdown in the substantia nigra/ventraltegmental area by synthetic small interfering RNA blocks the rewardingand locomotor effects of heroin. Neuroscience 158, 474-483Yi R, Pasolli HA, Landthaler M, Hafner M, Ojo T, Sheridan R, Sander C,O’Carroll D, Stoffel M, Tuschl T, Fuchs E. (2009) DGCR8-dependentmicroRNA biogenesis is essential for skin development. PNAS 106,498-502.Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T,Hafner M, Zavolan M, Tuschl T. (2008) Molecular characterization ofhuman Argonaute-containing ribonucleoprotein complexes and theirbound target mRNAs. RNA 14, 2580-2596.Landthaler M, Yalcin A, Tuschl T. (2004) The human DiGeorge syndromecritical region gene 8 and Its D. melanogaster homolog are required formiRNA biogenesis. Curr. Biol.14, 2162-7.Cardiovascular and Metabolic Disease Research 63