RNA recognition: controlling RNA-protein complexes with small molecules

Ramisetty, Sreenivasa Rao

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Description

Title

RNA recognition: controlling RNA-protein complexes with small molecules

Author(s)

Ramisetty, Sreenivasa Rao

Issue Date

2010-05-14T20:51:18Z

Director of Research (if dissertation) or Advisor (if thesis)

Baranger, Anne M.

Doctoral Committee Chair(s)

Baranger, Anne M.

Committee Member(s)

• Zimmerman, Steven C.
• Katzenellenbogen, John A.
• Hergenrother, Paul J.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Muscleblind-like protein (MBNL)
• Zinc finger (ZNF)

Abstract

ABSTRACT PART I. Investigation of Small Molecule Binding to an RNA Hairpin Loop Containing a Dangling End PART II. Unraveling the Interaction of Pathogenic RNAs with the MBNL1 Protein and Complex Inhibition by Small Molecules PART I. RNA plays important and versatile roles in gene expression by both carrying and regulating the information used to direct protein synthesis. Therefore, small molecules able to bind to RNA and alter these biological processes would be of great utility. This part of my thesis describes the virtual screening and identification of a quinoline derivative binding cooperatively to a GCAA RNA tetraloop containing a 3’ dangling end (tGCAA). The compound NSC5485 (QD2) was identified by performing a similarity search of the NCI database of 250,000 compounds and using the program AutoDock 3. Fluorescence and ITC experiments revealed that QD2 binds cooperatively to four identical binding sites on tGCAA RNA hairpin. The equilibrium binding dissociation constant of the four identical binding sites is 8.2 (±0.4) µM. CD spectroscopy and UV titration experiments suggested that binding of QD2 changes the conformation of RNA and perturbs the QD2 chromophore. PART II. Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including Huntington’s disease, Fragile X mental retardation, and myotonic dystrophy type 1. Myotonic dystrophy (DM1 and DM2) is an autosomal dominant neuromuscular disorder associated with a (CTG)n and (CCTG)n expansion in the 3’-untranslated region of the Dystrophia Myotonica protein kinase (DMPK) gene. The disease is characterized by a waning of the muscles (muscular dystrophy), eye-lens opacity and myotonia. The pathogenic poly(CUG)RNA and poly(CCUG)RNA binds to and sequesters key proteins, such as MBNL1 (muscleblind-like protein 1), preventing them from regulating proper splicing of different pre-mRNAs. The severity of disease correlates with the length of the repeat tract in peripheral blood. The first part of this project is about investigating the interaction of the MBNL1N protein with poly(CUG)RNA. We are interested in identifying important amino acids or zinc finger domains involved in recognition of MBNL1N protein to poly(CUG)RNA. To address this question we did alanine scanning for six amino acids and expressed truncated versions of the protein and studied their interaction with MBNL1N protein by gel-shift assays. In the second part, the inhibition of complexes formed between the toxic poly(CUG)RNA or poly(CCUG) RNA with MBNL1 protein by a small molecules has been shown by gel-shift assays. We identified small molecules containing triaminotriazine-acridine and triaminopyrimidine-acridine conjugates which can specifically inhibit (CUG)12 and (CCUG)6 complexes with MBN1N protein, respectively. Thus the compounds triaminotriazine-acridine and triaminopyrimidine-acridine conjugates are potential lead compounds for targeting DM1 and DM2, respectively.

Graduation Semester

2010-5

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Copyright 2010 Sreenivasa Rao Ramisetty

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