Advisory Committee Chair
David E Graves
Advisory Committee Members
Ching-Yi Chen
Bingdong Sha
Tim M Townes
Dmitry Vassylyev
Document Type
Dissertation
Date of Award
2012
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Nucleic acids are the storage of genetic information that needs to be expressed and inherited. As abundant cellular macromolecules they are also major players of a wide variety of biological processes. They usually undergo post-transcriptional modifications and/or fold into higher order structures in order to accomplish their distinct functions. Most biological events involving nucleic acids are achieved by proteins that interact with them. The functionality requires specific recognition of nucleic acids by proteins at molecular level. Nucleic acid chaperones are a group of nucleic acids binding proteins that associate with structured RNAs or DNAs and assist correct folding of their biological relevant conformations. Recent studies have shown that protein hnRNP A1 regulates the processing of the primary transcript of microRNA-18a. We demonstrated that hnRNP A1 recognizes the pri-miR18a through binding to its terminal loop which contains binding motifs recognized by two RRM domains of hnRNP A1 (UP1). We solved the crystal structure of UP1 in the complex with a single stranded RNA sequence derived from the terminal loop of pri-miR-18a, revealed the molecular basis for hnRNPA1-RNA recognition. Our result from mutagenesis and in vitro binding studies suggest chaperone activity of hnRNP A1 in the processing of pri-miRNA. Human telomeric DNA can form higher order structures called G-quadruplex which may be involved in telomere regulation. hnRNP A1 and its N-terminal region UP1 have been previously found bind and unfold this structure efficiently. Mutagenesis studies on the two conserved RRMs of UP1 demonstrated the distinct functions of RRM1 and RRM2; RRM1 is the key domain for initiating binding and unfolding of G-quadruplex structural motif; RRM2 contributes to the synergistic efficacy with RRM1 in the binding and unfolding process. We also identified the loop2 on the hTel22 G-quadruplex DNA as critical site for initiating recognition and unfolding by UP1. A model is proposed for how UP1 binds and unfolds telomeric G-quadruplex. Many small RNAs, including small-interfering RNAs and piRNAs in Drosophila, are methylated at 3' ends. The 3' end 2'-O-methylated RNA is preferably recognized by the PAZ domain of the Ago2. Here we present the crystal structure of the Drosophila Ago2 PAZ domain bound with a 3' end 2'-O-methylated RNA, providing a structural basis for the specific recognition of the methyl modification. In vitro binding and mutagenesis studies illustrate that 3' end methylated small RNAs are preferably bound by PAZ domains from Drosophila Ago2 and Piwi subfamily Argonaute proteins. We also characterized RNA chaperone activity of the human Ago1 PAZ domain, implying roles of PAZ domains in small RNA-target recognition.
Recommended Citation
Ding, Lei, "Structural And Molecular Studies Of Nucleic Acid Chaperones" (2012). All ETDs from UAB. 1525.
https://digitalcommons.library.uab.edu/etd-collection/1525
