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THE IRE RNA. |
Dr. Kathleen B. Hall
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Professor
Department of Biochemistry and Molecular Biophysics
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2nd Annual Hall Lab Pumpkin Glow!
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Director
Molecular Biophysics Program
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.
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Return to Washington University Biochemistry and Molecular Biophysics Homepage
Dr. Kathleen B. Hall (hall(at)biochem.wustl.edu)
Department of Biochemistry and
660
office: 314-362-4196
lab: 314-362-4197
or 314-747-8079
FAX: 314-362-7183
send email to kathleenhal(at)gmail.com
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