Stephen I Rudnick

University of Pennsylvania, Philadelphia, PA, United States

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Publications (5)31.13 Total impact

  • Stephen I. Rudnick · Veenu Aishwarya · Alan M. Gewirtz
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    ABSTRACT: Posttranscriptional gene silencing (PTGS) is a process by which a protein’s synthesis is impaired by targeting its messenger RNA (mRNA). The antisense oligodeoxynucleotide (AON) and RNA interference (RNAi) pathways can both accomplish PTGS by hybridization of a reverse complementary oligonucleotide and subsequent enzymatic degradation of the mRNA by an RNase mediated mechanism. We have investigated the influence of specific mRNA structural elements on short interfering RNA (siRNA) and AON targeting efficiency. Under identical conditions, both PTGS pathways are significantly inhibited by target mRNA secondary structure. Surprisingly, we found that an AON was less stringent in its requirement for mRNA target accessibility than the corresponding siRNA. By determining that the AON and siRNA guide strand have the same apparent K D in the absence of protein, we show that nucleic acid binding affinity does not explain their difference in in vivo silencing activity. Rather, it appears that RISC must increase the binding affinity of the siRNA for the target. Furthermore, RNA binding proteins are also potent inhibitors of AON activity. We conclude that mRNA secondary and quaternary structure play important roles in PTGS by significantly affecting the ability of a siRNA or AON to hybridize with their intended target. Recognition of these effects will facilitate the design of more efficient antisense molecules for therapeutically motivated gene silencing and argue for continued mechanistic studies on AON and siRNA mediated mRNA destruction. KeywordsRNA structure-siRNA-Antisense-Oligodeoxynucleotide-Gene silencing-RNA cleavage
    09/2010: pages 255-275;
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    ABSTRACT: GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4(+) cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4(+) T cells, decreased expression of GATA-3 and Th2 cytokines. In primary T cells, c-Myb allows GATA-3 to autoactivate its own expression, an event that requires the direct interaction of c-Myb and GATA-3 on their respective binding sites in promoter of GATA-3. Immunoprecipitation revealed that the c-Myb/GATA-3 complex contained Menin and mixed lineage leukemia (MLL). MLL recruitment into the c-Myb-GATA-3-Menin complex was associated with the formation Th2 memory cells. That MLL-driven epigenetic changes were mechanistically important for this transition was suggested by the fact that silencing c-Myb significantly decreased the methylation of histone H3K4 and the acetylation of histone H3K9 at the GATA-3 locus in developing Th2 and CD4(+) effector/memory cells. Therefore, c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and, with the recruitment of MLL, Th2 cell maturation in primary human peripheral blood T cells.
    Blood 08/2010; 116(8):1280-90. DOI:10.1182/blood-2009-05-223255 · 10.45 Impact Factor
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    ABSTRACT: Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON- vs. siRNA-mediated PTGS, AON- and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was approximately 3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nucleotides, than a corresponding siRNA. Nonetheless, in vivo, siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo. We used a well defined RNA-binding protein, alphaCP, and its corresponding high-affinity RNA-binding site to explore this hypothesis. We found that prebound alphaCP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of alphaCP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo.
    Proceedings of the National Academy of Sciences 10/2008; 105(37):13787-92. DOI:10.1073/pnas.0805781105 · 9.67 Impact Factor
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    ABSTRACT: Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.
    Molecular and Cellular Biology 04/2007; 27(6):2048-58. DOI:10.1128/MCB.01100-06 · 4.78 Impact Factor
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    Stephen I. Rudnick · Alan M. Gewirtz
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    ABSTRACT: Molecular Therapy (2006) 13, S276|[ndash]|S276; doi: 10.1016/j.ymthe.2006.08.795 716. Differential Effects of Secondary Structure on Antisense Oligonucleotide (AON) and Short Interfering RNA (siRNA) Mediated mRNA Cleavage Stephen I. Rudnick1 and Alan M. Gewirtz21Department of Pharmacology, University of Pennsylvania, Philadelphia, PA2Department of Medicine, University of Pennsylvania, Philadelphia, PA
    Molecular Therapy 04/2006; 13:S276-S276. DOI:10.1016/j.ymthe.2006.08.795 · 6.23 Impact Factor