國立高雄大學圖資館 |

Language: English

Back

Alteration of gene expression by specially designed RNA decoys

Record Type:	Electronic resources : Monograph/item
Title/Author:	Alteration of gene expression by specially designed RNA decoys
Author:	Eastmond, Dawn L.
Description:	130 p.
Notes:	Source: Dissertation Abstracts International, Volume: 65-03, Section: B, page: 1305.
Notes:	Supervisor: Stephen Liebhaber.
Contained By:	Dissertation Abstracts International65-03B.
Subject:	Chemistry, Biochemistry.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3125809
ISBN:	0496730983

Alteration of gene expression by specially designed RNA decoys
Eastmond, Dawn L.

Alteration of gene expression by specially designed RNA decoys [electronic resource] - 130 p.

Source: Dissertation Abstracts International, Volume: 65-03, Section: B, page: 1305.

Thesis (Ph.D.)--University of Pennsylvania, 2004.

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression including mRNA processing, stabilization, sublocalization, and translation. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH domain RNA-binding proteins. These polyC-binding proteins have been implicated in a wide spectrum of post-transcriptional controls. In the first part of the study, three categories of RNA decoys to alphaCPs were studied: polyC homopolymers, native mRNA binding sites, and a high-affinity sequence selected from a combinatorial library. Since alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear and cytoplasmic RNA frameworks. The ability of the resultant chimeric decoy transcripts to bind alphaCP was monitored and quantified by RNA-EMSA; their bioactivity was monitored both in vitro and in transfected cells by inhibition of the well characterized alphaCP-dependent translational activity of the polio viral mRNA internal ribosome entry site (IRES). A subset of decoy transcripts was identified that effectively inhibit alphaCP-dependent translation in intact cells. In the second phase of the study, we applied these same decoy RNAs to target halpha-globin mRNA stability. The stability of halpha-globin mRNA was studied in MEL cells that stably expressed the tet-transactivator (tTA) protein; in these cells the halpha-globin gene, under tet-Operator control, could be conditionally expressed over a short (4 hour) window. Halpha-globin mRNA stability was determined by sequential assays of halpha-globin mRNA content in these cells after the transcriptional pulse. The tet-O/halpha-globin gene was transfected into the MEL/tTA cells either alone or in combination with plasmids expressing the most effective alphaCP decoys. We observed a dose-dependent destabilization of halpha-globin mRNA in the presence of both nuclear and cytoplasmic decoy transcripts. Control framework transcripts lacking decoy cassettes had no effect on stability. Furthermore, alpha-globin mRNAs that were already destabilized due to mutation or deletion of the alphaCP binding site in their 3'UTRs were unaffected by active decoy transcripts. These data confirm the central importance of alphaCP to the stabilization of halpha-globin mRNA and establish the feasibility of triggering incremental decreases in halpha-globin gene expression by decoy-mediated mRNA destabilization. Such destabilization would be predicted to modify the severity of beta-thalassemia by diminishing the accumulation of alpha-globin, which is toxic to the developing red cell.

ISBN: 0496730983Subjects--Topical Terms:

226900
Chemistry, Biochemistry.

Alteration of gene expression by specially designed RNA decoys
LDR:03830nmm _2200265 _450 001 162538
005 20051017073457.5
008 230606s2004 eng d
020 $a 0496730983
035 $a 00149039
035 $a 162538
040 $a UnM $c UnM
100 0 $a Eastmond, Dawn L. $3 227680
245 1 0 $a Alteration of gene expression by specially designed RNA decoys $h [electronic resource]
300 $a 130 p.
500 $a Source: Dissertation Abstracts International, Volume: 65-03, Section: B, page: 1305.
500 $a Supervisor: Stephen Liebhaber.
502 $a Thesis (Ph.D.)--University of Pennsylvania, 2004.
520 # $a RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression including mRNA processing, stabilization, sublocalization, and translation. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH domain RNA-binding proteins. These polyC-binding proteins have been implicated in a wide spectrum of post-transcriptional controls. In the first part of the study, three categories of RNA decoys to alphaCPs were studied: polyC homopolymers, native mRNA binding sites, and a high-affinity sequence selected from a combinatorial library. Since alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear and cytoplasmic RNA frameworks. The ability of the resultant chimeric decoy transcripts to bind alphaCP was monitored and quantified by RNA-EMSA; their bioactivity was monitored both in vitro and in transfected cells by inhibition of the well characterized alphaCP-dependent translational activity of the polio viral mRNA internal ribosome entry site (IRES). A subset of decoy transcripts was identified that effectively inhibit alphaCP-dependent translation in intact cells. In the second phase of the study, we applied these same decoy RNAs to target halpha-globin mRNA stability. The stability of halpha-globin mRNA was studied in MEL cells that stably expressed the tet-transactivator (tTA) protein; in these cells the halpha-globin gene, under tet-Operator control, could be conditionally expressed over a short (4 hour) window. Halpha-globin mRNA stability was determined by sequential assays of halpha-globin mRNA content in these cells after the transcriptional pulse. The tet-O/halpha-globin gene was transfected into the MEL/tTA cells either alone or in combination with plasmids expressing the most effective alphaCP decoys. We observed a dose-dependent destabilization of halpha-globin mRNA in the presence of both nuclear and cytoplasmic decoy transcripts. Control framework transcripts lacking decoy cassettes had no effect on stability. Furthermore, alpha-globin mRNAs that were already destabilized due to mutation or deletion of the alphaCP binding site in their 3'UTRs were unaffected by active decoy transcripts. These data confirm the central importance of alphaCP to the stabilization of halpha-globin mRNA and establish the feasibility of triggering incremental decreases in halpha-globin gene expression by decoy-mediated mRNA destabilization. Such destabilization would be predicted to modify the severity of beta-thalassemia by diminishing the accumulation of alpha-globin, which is toxic to the developing red cell.
590 $a School code: 0175.
650 # 0 $a Chemistry, Biochemistry. $3 226900
690 $a 0487
710 0 # $a University of Pennsylvania. $3 212781
773 0 # $g 65-03B. $t Dissertation Abstracts International
790 $a 0175
790 1 0 $a Liebhaber, Stephen, $e advisor
791 $a Ph.D.
792 $a 2004
856 4 0 $u http://libsw.nuk.edu.tw/login?url=http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3125809 $z http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3125809