Apart from carrying genetic information, RNAs also act as effectors of cellular processes through folding into intricate secondary and tertiary structures. The ubiquitous RNA structures in eukaryotic mRNAs, in collaboration with specific RNA-binding proteins, control many aspects of the post-transcriptional regulation of gene expression. However, the molecular bases for the recognition of these mRNA structures by their protein partners remain poorly understood due to the lack of structural information. This dissertation presents our structural studies on two protein-RNA complexes that both include regulatory mRNA stem-loop structures. We first describe the crystal structure of a ternary complex including the highly conserved human histone mRNA stem-loop (SL), the stem-loop binding protein (SLBP) and the 3′ to 5′ exonuclease 3′hExo. This structure identifies a single sequence-specific interaction between the SL and SLBP, and the mostly shape-dependent RNA-recognition mode by both proteins. In addition to explaining the large body of biochemical and biophysical data on this complex accumulated over the last two decades, we also for the first time elucidate the induced-fit mechanism underlying the cooperativity between SLBP and 3′hExo. We next shift our focus to a class of less conserved mRNA stem-loop structures named constitutive decay elements (CDE). The RNA-binding ROQ domain of Roquin recognizes the various CDEs and mediates the decay of CDE-containing mRNAs, which predominantly encode proteins responsible for inflammation and autoimmunity. Structural and biochemical studies of the ROQ domain in complex with two different CDE RNAs unexpectedly reveal two distinct RNA binding sites on this protein, one recognizing CDE stem-loops and the other binding to double-stranded RNAs. The stuctures are also in agreement with the versatility of Roquin and have opened up new avenues to investigating its functions in modulating the stability of target mRNAs.

[Truncated abstract] Regulation of mRNA stability is an important posttranscriptional mechanism involved in the control of gene expression. The rate of mRNA decay can differ greatly from one mRNA to another and may be regulated by RNA-protein interactions. A key determinant of mRNA decay are sequence instability (cis) elements often located in the 3' untranslated region (UTR) of many mRNAs. For example, the AU rich elements (AREs), are such well characterized elements, and most commonly involved in promoting mRNA degradation, and specific binding of proteins to these elements leading to the stabilization of some mRNAs. Other cis-elements have been described for mRNA in which mRNA stability is a critical component of gene regulation. This includes the androgen receptor (AR) UC-rich cis element in its 3'UTR. The AR is a key target for therapeutics in human prostate cancer and thus understanding the mechanism involved in regulating its expression is an important goal. The [alpha]CP1 protein, a KH-domain containing RNA-binding protein has been found to bind this UC-rich region of the AR and is thought to play an important role in regulating AR mRNA expression. [alpha]CP1 protein is a triple KH (hnRNP K homology) domain protein with specificity for Crich tracts of RNA and ssDNA (single stranded DNA). Relatively little is known about the structural interaction of [alpha]CP1 with target RNA cis elements, thus the present study aimed to better understand the nature of interaction between 30 nt 3'UTR UC-rich AR mRNA and [alpha]CP1 protein using various biophysical techniques, in an attempt to determine which [alpha]CP1 domain or combination of domains is involved in RNA-binding. These studies could ultimately provide novel targets for drugs aimed to regulate AR mRNA expression in prostate cancer cells. At the commencement of this study little was known about the structure of the [alpha]CP1- KH domains and their basis for poly (C) binding specificity. ... Additional studies addressed the significance of the four core recognition nucleotides (TCCC) using a series of cytosine to thymine mutants. The findings verified some of the results predicted from structural studies, especially the need for maximum KH binding to a core tetranucleotide recognition sequence. Our mutational studies of the four core bases confirmed the importance of cytosine in positions two and three as no binding was observed, while some binding was observed when the fourth base was mutated. In summary, the work presented in this thesis provides new detailed insight into the molecular interactions between the [alpha]CP1-KH domain and AR mRNA. Furthermore, these studies shed light on the nature of protein/mRNA interactions in general, as well as the specific complex that forms on AR mRNA. These studies have provided new understanding into the mode of [alpha]CP1 binding at a target oligonucleotide binding site and, provide a foundation for future studies to define structure of multiprotein/oligonucleotide complexes involved in AR mRNA gene regulation. Understanding the detailed interaction between the AR mRNA and [alpha]CP1 could provide possible targets for drug development at reducing AR expression in prostate cancer cells by interfering with the interaction of [alpha]CP1 and AR-mRNA.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Hfq is a bacterial RNA-binding protein recently shown to contain the Sm motif, a characteristic of Sm proteins that function in RNA processing in archaea and eukaryotes. Hfq plays a major role in RNA-RNA interactions regulating translation. Comparative structural modeling and amino acid sequence alignment were used to predict the 3-D structure of Hfq and the model was in excellent agreement with the crystal structure which determined for S. aureus Hfq. The evolution of Hfq was explored by a BLAST search of microbial genomes followed by phyletic analysis. About half of the genomes examined contain at least one gene coding for Hfq. The presence and absence of Hfq closely followed major bacterial clades. The potential RNA binding residues on the two surfaces of the Hfq hexamer were proposed based on the bioinformatics studies and the mutant Hfq proteins with either single or double mutations on the two surfaces of the Hfq hexamer were generated. Their RNA binding properties was biophysically studied by gel-shift assay, fluorescence anisotropy and fluorescence quenching techniques. Results indicated that 1) point mutations on the distal surface of the Hfq hexamer, Y25A and K31A, have a major effect on A18 binding. Both reduce binding by about 1000 fold. Mutations on the proximal surface have a small or no influence on A18 binding. 2) Two mutations, F39A and R16A, on the proximal surface of the Hfq structure reduce binding to the DsrA domain II by 10 fold. Other mutations reduce binding by less than 2 fold. 3) An amino acid covariance was observed in L12 and F39. Mutation L12F can partially restore F39A in DsrA RNA binding. 4) It appears that two Hfq hexamers cooperatively bind one RNA for both DsrADII and A18.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biochemistry. Title from pdf file main screen (viewed on February 12, 2010). Includes bibliographical references.

Title from title page of PDF (University of Missouri--St. Louis, viewed February 22, 2010). Includes bibliographical references.

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<p>RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including &#967;) and 12 non-hydrogen atoms, compared to 4 torsions (including &#967;1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex. </p><p>My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures, a method for assigning RNA backbone structure based on NOE data was developed, leading to improved identification and building of RNA backbone conformation in NMR ensembles. For crystallography, our method of diagnosing the correct ribose pucker from clear observables allows reliable assessment of pucker in validation or refinement. Observed differences in bond-lengths, bond-angles, and dihedrals have been categorized by sugar pucker in the PHENIX refinement package. I have shown that this improves the refinement behavior of both pucker and geometry. </p><p>There have also been improvements in identifying structural motifs. Many previously identified structural motifs have now been defined in terms of backbone suitestrings, a series of 2-character code divisions of RNA backbone that show the best clustering of dihedral angle correlations. Combined with a BLAST-like alignment program called SuiteAlign, these suitestrings were quickly and easily identified in a number of structures, eventually leading to the discovery of multiple instances of T&#968;C-loop structures in the ribosome.</p><p>To facilitate error diagnosis and corrections in RNA-protein complexes, as well as to expand the knowledge base of the scientific community as a whole, a database of RNA-protein interaction motifs has been developed. This database is rooted in the quality-filtering, visualization, and analysis techniques of the Richardson lab, particularly those developed by Laura Murray specifically for RNA structures.</p><p>The consensus backbone conformers, pucker diagnosis, and all-atom contacts have been combined to develop first manual and then automated tools for RNA structure correction. I have applied all these techniques to improve the accuracy of a number of important RNA and RNA/protein complex structures.</p> / Dissertation