Structure, Dynamics and Functional Mechanisms of the PWI Domain and the RNA-Binding Domain of Rotavirus Non-Structural Protein 3

Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Dr. Blair Szymczyna

Second Advisor

Dr. David Huffman

Third Advisor

Dr. Ramakrishna Guda

Fourth Advisor

Dr. Karim Essani


RNA binding proteins play essential roles in many cellular processes, including structural, regulatory and catalytic roles. The molecular mechanisms by which many RNA-binding proteins recognize and interact with their targets are still unknown, and remain the subject of extensive research. The structural and functional properties of the rotavirus Non-structural Protein 3 (NSP3) and the PWI RNA binding domains are described in this study.

Rotavirus is a member of the Reoviridae family of viruses and can cause severe, life threatening diarrhea in children under the age of 5. The Rotaviruses genome consists of 11 segments of double-stranded RNA, which encodes for 11-12 different proteins. Six non-structural proteins (NSPs) play regulatory roles in the replication cycle of the virus. Non-structural protein 3 (NSP3) is believed to regulate virus protein synthesis by hijacking host translation machinery and promoting viral mRNA translation using a mechanism that is analogous to Poly-Adenosine Binding Protein. The N-terminal, RNA-binding domain of NSP3 binds a conserved tetranucleotide sequence at the 3’ end (---GACC) of rotavirus RNA molecules, while the C-terminal domain binds host translation initiation factor eIF4G. Gene suppression studies recently revealed that NSP3 also prevents the expression of host mRNA by preventing export from the cell nucleus using an unknown mechanism. The relative importance and the molecular mechanisms of NSP3 functions are still controversial. The aims of the study were to discover the structural features and functional mechanisms involved in NSP3 binding to RNA, and to elucidate the role of RNA binding in different protein functions. NMR spectroscopic studies of several NSP3 protein constructs revealed that the RNA binding domain contains three structurally distinct sub-domains that “hug” the RNA target during the binding mechanism.

The PWI domain is named for a three amino acid residue sequence, Proline(P)- Tryptophan(W)-Isoleucine(I), that is highly conserved, and found in several proteins involved in pre-messenger RNA (pre-mRNA) processing. Pre-mRNA processing is essential in the generation of mRNA molecules that can be exported from the nucleus and translated into proteins, and can result in the formation of many different proteins from a single gene. Incorrect regulation of processing is associated with several diseases, including cancer and heart failure. The PWI domain consists of a PWI motif and an adjacent basic region that is critical for the domain’s nucleic acid binding properties. The aim of the study was to investigate the mechanism by which the PWI motif and the adjacent basic region cooperatively bind to nucleic acids. Several biophysical approaches were used to investigate the nucleic acid binding mechanism of PWI domains from different proteins. Fluorescence spectroscopy was used to assess binding stoichiometry, while mobility shift assays measured binding affinity and cooperativity. PWI domains from PRP3, SRm160 and RBM25 are found to bind nucleic acids with a low micromolar binding affinity, and two or more proteins are involved in complex formation. NMR spectroscopy revealed the structural and conformationally dynamic properties of the domain.


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