THE IRE RNA.
Dr. Kathleen B. Hall
Department of Biochemistry and Molecular Biophysics
RNA and RNA:Protein interactions provide biological regulation at all levels of cellular function. We study RNA molecules and their interactions with protein partners in order to understand their biology. We use biochemistry, molecular biology, and molecular biophysics to understand the properties of these molecules.
Which RNAs do we study?
The human Iron Response Element (IRE) RNA hairpin is illustrated above. Its six nucleotide loop is best characterized as floppy; there is no one structure that describes it. The loop nucleotides are phylogenetically conserved, and are recognized by the IRE Binding Protein. We have studied the dynamics of the loop nucleotides, to determine the timescale of motions, their amplitudes, and their correlations. In addition to providing a picture of what this RNA could look like in solution, the IRE has served as a model system for us to study RNA flexibility. We have used steady-state and time-resolved fluorescence, NMR relaxation, and molecular dynamics simulations to describe this RNA.
Recently, we have begun to explore the structures adopted by the Mg2+-dependent riboswitch (Chromie, M.J., Shi, Y., Latifi, T., Groisman, E.A. 2006. An RNA sensor for intracellular Mg2+. Cell 125 : 71-84.)Cell). Riboswitches are RNA sequences, typically found in the 5’ untranslated regions (UTR) of bacterial mRNAs, which undergo ligand-dependent conformational changes to control transcription or translation of the message. The Mg2+-dependent riboswitch regulates transcription of the mgtA gene. The mgtA gene codes for the Mg2+-transporter A protein, which transports Mg2+ into the cell; transcription of its mRNA is controlled by intracellular Mg2+ levels. Originally found in Salmonella enterica, the riboswitch has been identified in E. coli, Klebsiella, and Citrobacter. Since RNA structures rely on Mg2+ ions for adopting correct tertiary structures, the discovery of an RNA that uses Mg2+ as a regulatory cofactor is a striking evolutionary adaptation. The mechanism it employs to effect transcription termination is unknown, and that is what we hope to discover.
Which RNA:Protein interactions do we study?
Our work has focused for some time on human U1A protein and its recognition of two RNAs. In the U1 snRNP, U1A is bound to Stemloop II of the human U1 snRNA, where it uses seven out of ten nucleotides in the loop for specific recognition. In the nucleus, U1A also binds to its own pre-mRNA, in an interaction with the Polyadenylation Inhibition Element (PIE) RNA. This structure, found at the 3' untranslated region of the U1A pre-mRNA, is bound by two U1A molecules to block adenylation. Only the N-terminal RNA Binding Domain (RBD) or RNA Recognition Motif (RRM) of the protein binds RNA; the function of the C-terminal RRM remains unknown.
The Drosophila SNF protein is an evolutionary off-shoot of U1A. The fly doesn't have U1A protein, and it also lacks the related U2B" protein. Instead, it has SNF (sans fille), which appears to be a naturally occurring RRM chimera formed from U1A and U2B”. It binds both the U1 snRNA Stemloop II and the U2 SNRNA Stemloop IV, but its affinity for the U1 RNA is far weaker than its U1A predecessor. What has happened to this protein such that it binds both RNAs with weak affinity? This project is in its infancy.
In contrast to the U1A protein, which binds with exquisite specificity and high affinity to Stemloop II, the human polypyrimidine tract binding protein (PTB) binds to (U/C)n tracts. In the nucleus, it participates in regulation of alternative splicing, where its most common role is exon repression, i.e. exclusion of an exon from spliced mRNA. PTB binds to polypyrimidine sequences within both introns and exons, usually proximal to the 3’ splice site of pre-mRNAs, but the locations, lengths, and numbers of polypyrimidine tracts vary enormously among pre-mRNA sequences. The mechanisms by which PTB excludes an exon, and conversely, how the exclusion is relieved, are obscure.
The current model of PTB binding to its pre-mRNA targets is that it forms a multimeric PTB:RNA complex. Our in vitro experiments have shown that within these complexes, there are different classes of bound PTB proteins: some directly contact the RNA, while others associate through protein:protein interactions. We propose that the assembly state of a PTB:RNA complex determines the fate of the exon by either permitting access to the RNA by other regulatory proteins when only a few PTB molecules are directly bound, or excluding access when the complexes are fully formed. From our quantitative description of the conformation and composition of these PTB:RNA complexes, a mechanism of exon exclusion by PTB will be developed.
For more information on these projects, please see our Lab Projects page.
Dr. Kathleen B. Hall (kathleenhal(at)gmail.com)
Department of Biochemistry and
Last Update: October 30, 2009
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