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" פיתוח אולגונוקלאוטידים ממסכים כמעכבי פקטורי שחבור ופעילותם כמדכאי סרטן "
Denichenko, Polina
Karni, Rotem
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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1112324
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TLpq2526090040
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| Main Entry
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Denichenko, Polina
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Karni, Rotem
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| Title & Author
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פיתוח אולגונוקלאוטידים ממסכים כמעכבי פקטורי שחבור ופעילותם כמדכאי סרטן\ Denichenko, PolinaKarni, Rotem
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| College
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The Hebrew University of Jerusalem (Israel)
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| Date
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2019
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| student score
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2019
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| Degree
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Ph.D.
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| Page No
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101
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| Abstract
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Splicing factors bind, in most cases, to a degenerate motif in the pre-mRNA of the target gene and either recruit or repel the spliceosome to/from nearby splice sites. Many splicing factors possess RNA-independent functions, such as protein-protein interactions in cellular complexes, which are essential for proper cellular functions. The involvement of splicing factors in multiple diseases and processes was the driving force behind the idea to develop splicing factor specific inhibitors. Inhibition of splicing factor expression by available techniques, such as siRNAs or antisense oligonucleotides, could have broad detrimental effects on cell fate. Therefore, we sought to develop an efficient splicing factor inhibitor which will target only the splicing activity of the factor, without interfering with its other activities. In this work we designed small modified RNA oligonucleotides containing several repeats of a specific RNA motif. These RNA oligonucleotides can bind to the RRM domain of a specific splicing factor and, by serving as decoy molecules, can inhibit the splicing activity of a specific splicing factor. In order to test the feasibility of this approach we chose to target three alternative splicing factors; RBFOX1/2, PTBP1 and SRSF1. RBFOX1 and RBFOX2 are members of a splicing factor family that regulates hundreds of splicing events as determined by direct RNA binding using RNA CLIP and RNA-seq experiments and are known to be involved in multiple diseases. Most splicing factors bind degenerate motifs in the pre-mRNA of their target genes. An exception is RBFOX1/2 proteins that bind a well-defined motif (U)GCAUG, which makes it an ideal target for specific inhibition. PTBP1 and SRSF1 are two splicing factors known to be involved in cancer. Here we provide a proof-of-principle demonstration that RNA decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1 can inhibit their splicing activity. We show that decoy oligonucleotides against RBFOX1/2 proteins can specifically bind RBFOX1/2 and inhibit their splicing activity, as measured by changes in splicing of known targets in vitro. We evaluated the number of motif repeats needed for effective decoy activity. Using these oligonucleotides in a zebrafish model of muscle development, we demonstrate altered splicing of known muscle related targets and impaired muscle development. Decoy oligonucleotides against PTBP1 showed specific binding to the protein and inhibition of its splicing activity in vitro. Inhibition of oncogenic properties of breast and glioblastoma cancer cells was determined after transfection of decoy oligonucleotides using soft agar, clonogenic and proliferation assays. The decoy oligonucleotides against the oncoprotein SRSF1 inhibited both its splicing activity, as well as, the oncogenic properties of breast and glioblastoma cancer cells. We determined this occur via activation of the p38-MAPK stress pathway. Injection of glioblastoma cells transfected with decoy oligonucleotides against SRSF1 into mice brains formed smaller tumors compared to the control oligonucleotides. The decoy oligonucleotides target only the splicing activity of the splicing factor and can act directly and immediately, independent of any cellular component. This is in contrast to siRNAs or antisense oligonucleotides, which are dependent on the half-life of the factor and the integrity of cellular components (e.g. RNAi machinery). Thus, decoy oligonucleotides present a novel methodology to specifically downregulate splicing factor activity. These molecules have the potential to be used to treat diseases where splicing factors are up-regulated.
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Adenosine
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Amino acids
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Binding sites
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Genotype phenotype
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Heart failure
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Metastasis
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