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We are interested in understanding post-transcriptional regulation of gene expression in human cells. Our studies focus on the role of siRNAs, miRNAs and piRNAs in silencing of genes, with the aim of understanding their natural function, as well as their applications as a research tool and therapeutic for treatment of genetic disease.

Biochemical analysis of the mechanism of RNA interference in human cells

Small RNA profiling of human cell types in normal and disease states and analysis of miRNA function

siRNA user guide

Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of sequence-specific gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms (Fig. 1). Our laboratory investigates the mechanisms of RNA silencing in mammalian and human systems and catalogues the natural sources of dsRNA (such as miRNAs, repetitive gene sequences and viruses) contributing to mammalian gene regulation. The understanding of RNA silencing mechanisms in human has important implications for exploring its links to genetic diseases as well as the development of siRNA-based silencing applications and therapeutic agents.
We develop biochemical and cell-based assays to dissect the mechanism of siRNA-guided mRNA degradation, miRNA-guided translational repression, and repeat-associated siRNA-guided chromatin modification. New factors involved in RNA silencing are being identified and the order of assembly of functional complexes is being investigated.
Our most productive approach for identifying new components of the RNA silencing machinery is the native purification of RNPs containing known components of the RNA silencing machinery. In general, we establish stable cell lines expressing epitope-tagged versions of these proteins, such as the family of Argonaute proteins, or type III RNases, and grow these cell lines in large bioreactors to obtain sufficient material for proteomic analysis of the complexes of various size and composition. To gain a clear understanding of the RNA-protein interaction network, we clone and sequence the bound small RNAs and target mRNAs for particular RNPs. As an alternative strategy to our protein affinity purification approach, we have also developed sequence-specific RNP purification protocols using biotinylated antisense oligonucleotides to isolate proteins associated with particular small RNAs. This protocol can also be applied after protein affinity purification to characterize specific sub-complexes.
To obtain a deeper mechanistic understanding of the factors identified in these purifications, we use biochemical assays that reconstitute complex RNP assembly, and small RNA-directed cleavage activity, translational repression or chromatin modification. The specific depletion of proteins prior to reconstitution assays is either accomplished by using antibody-based immuno-depletion or by preparing extracts from cells treated with siRNAs cognate to the target gene. The reconstitution is carried out using recombinantly expressed protein in bacterial or insect cell expression systems.
To dissect RNA silencing pathways in living cells, we have established stable cell lines that carry reporter genes under the control of RNA silencing processes. Our cell-based reporter assays measure activation of reporter gene expression, which occurs upon interference with the RNA silencing machinery. In the past, interference with RNA silencing was recorded as reduction of reporter gene expression, which could be complicated by secondary effects such as reduced cell viability or proliferation. Our positive readout systems are less prone to artifacts and easier to analyze because of the absence of reporter background expression. These cell lines will be used for high-throughput screening to identify small molecule inhibitors of RNA silencing pathways and in siRNA-based genome-wide screens to identify new candidate genes required for RNA silencing.

References

A. Aravin, D. Gaidatzis, S. Pfeffer, M. Lagos-Quintana, P. Landgraf, N. Iovino, P. Morris, M. J. Brownstein, S. Kuramochi-Miyagawa, T. Nakano, M. Chien, J. J. Russo, J. Ju, R. Sheridan, C. Sander, M. Zavolan, T. Tuschl,
A novel class of small RNAs bind to MILI protein in mouse testes, Nature, 2006, 442, 203-207.

G. Meister, M. Landthaler, L. Peters, J. Chen, H. Urlaub, R. LŸhrmann, T. Tuschl,
Identification of novel Argonaute-associated proteins, Curr. Biol. 2005, 15, 2149-2155.

M. Landthaler, A. Yalcin, T. Tuschl,
The human DiGeorge Syndrome Critical Region Gene 8 and its D. melanogaster homolog are required for miRNA biogenesis, Curr. Biol, 2004, 14, 2162-2167.

G. Meister, M. Landthaler, A. Patkaniowska, Y. Dorsett, G. Teng, T. Tuschl,
Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs, Mol. Cell, 2004, 15, 185-197.

J. Martinez, A. Patkaniowska, H. Urlaub, R. Lührmann, T. Tuschl,
Single-stranded antisense siRNAs guide target RNA cleavage in RNAi, Cell, 2002, 110, 563-574.

M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl,
Identification of novel genes coding for small expressed RNAs, Science, 2001, 294, 853-858.

S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, Klaus Weber, T. Tuschl,
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001, 411, 494-498.

S. M. Elbashir, W. Lendeckel, T. Tuschl,
RNA interference is mediated by 21 and 22 nt RNAs, Genes Dev., 2001, 15, 188-200.



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To understand the biology of RNA silencing, we identify dsRNA expression units in all cell types as a function of development and disease state, including viral infection. In collaboration with computational biologists Mihaela Zavolan (University of Basel, Switzerland) and Chris Sander (Memorial Sloan-Kettering Cancer Center) and with James Russo (Columbia University Genome Center), we now prepare, sequence, and analyze large numbers of small RNA libraries. Although it is possible to use miRNA microarrays, novel miRNA genes are still being discovered and thus would not be included in such an analysis. In addition, rare siRNAs originating from overlapping transcripts or regions of heterochromatin would also go undetected.
To date, we have recorded small-RNA profiles for about 250 different samples, including tissues of mouse, rat, and human, and many cell lines derived from human tumors and patient material. We have identified miRNAs that are highly tissue- and cell-type specific. Some miRNAs appear to be turned on as a function of development, and some appear to play a role in controlling cell proliferation.
We also discovered miRNAs expressed from the herpesvirus family, including Epstein-Barr and Kaposi sarcoma virus. This was a surprising finding because it was often speculated that RNAi in mammals is part of an antiviral mechanism rather than part of the viral regulatory repertoire for specific manipulation of host and viral gene expression. We are continuing to catalogue the small RNAs expressed from other human viruses.
Tissue- and cell-type-specific miRNA and siRNA profiles provide critical information for interpreting cDNA and proteomic data that are or will become available. The small RNA profiles will make it easier to identify correlations between dysregulation of dsRNA expression units (miRNAs, sense/antisense-overlapping transcripts) and protein expression in disease states. These investigations will help clarify whether a disease originates from dysregulation of a small noncoding RNA or from mutations within the small RNA or its target that perturb their interaction.
In animals, miRNAs regulate many different biological processes, including cell-lineage specification, apoptosis, neuronal development, cholesterol metabolism, and hormonal secretion. The vast majority of the several hundred mammalian miRNAs have no known function.
We use several approaches to identify and validate targets and function of miRNAs. The first approach relies on genome-wide predictions of miRNA targets based on sequence similarity and evolutionary cross-species conservation (in collaboration with the Zavolan and Sander labs). As our mechanistic understanding of miRNA-guided repression and its sequence specificity improves, rules for the target predictions will be refined. In collaboration with Markus Stoffel (Rockefeller University/ETH Zuerich), we use nuclease-resistant 2'-O-methyl antisense oligoribonucleotides (antagomirs) to inhibit miRNAs sequence-specifically in mice. We also collaborate with Arndt Borkhardt (Pediatric Hematology & Oncology, LMU and von Haunersches Children's Hospital, Munich) and Marc van de Vijver (Department of Pathology, Netherlands Cancer Institute) to examine the role of miRNAs in cancers.
As an alternative to the bioinformatic-driven approach, we develop biochemical means to isolate miRNAŠtarget-mRNA complexes directly. Together with the determination of the spatial and temporal expression of miRNAs and their target mRNAs, these approaches will contribute to the elucidation of the biological function of miRNA regulatory networks.
Single-nucleotide changes as well as micro and macro deletions and/or insertions can abrogate, strengthen, or weaken miRNAŠtarget RNA interactions. As our understanding of miRNA regulation and function progresses, we will initiate genotyping studies to examine the association of miRNA- and mRNA-targeting-site sequence variation with heritable diseases or predisposition to diseases. The targeting sites for miRNAs are predominantly located outside of the protein-coding region in the 3'-untranslated region of target mRNAs, a region that has received little attention in disease-related mutational analysis. Perturbation of the conserved network of miRNA and target mRNA interactions may offer new explanations for idiopathic diseases.

References

T. Tuschl, P. A. Sharp, D. P. Bartel,
A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation, RNA, 2001, 7, 29-43.

C. B. Burge, T. Tuschl, P. A. Sharp,
Splicing of precursors to mRNAs by the spliceosomes: in The RNA World 2nd edition, R. F. Gesteland, T. R. Cech, J. F. Atkins (eds.), Cold Spring Harbor Laborotory Press, Cold Spring Harbor, NY, 1999, 525-560.

T. Tuschl, P. A. Sharp, D. P. Bartel,
Selection in vitro of novel ribozymes from a partially randomized U2 and U6 snRNA library, EMBO, 1998, 17, 2637-2650.

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