Since the genetic code was elucidated in the early 1960's, it has been assumed that mRNAs are always decoded in three base codons, that any deviation from this fundamental rule must be erroneous and thus, deleterious. However, over the past decade, we have shown that a significant fraction of cellular mRNAs harbor cis-acting sequence elements that direct elongating ribosomes to shift reading frame by one base in the 5' (-1) direction. In cellular mRNAs, such Programmed -1 Ribosomal Frameshift (-1 PRF) signals direct ribosomes to premature termination codons where they become substrates for rapid degradation through the Nonsense-Mediated mRNA Decay (NMD) pathway, resulting in decreased expression of the proteins encoded by these mRNAs. Importantly, rates of mRNA degradation are proportional to rates of -1 PRF, a relationship that is conserved in eukaryotes from yeast to humans. Observations from yeast to humans that global changes in -1 PRF rates are deleterious to cellular function suggested that regulation of -1 PRF must be sequence- specific. Recently, we discovered that this is achieved through the interactions between -1 PRF signals and miRNAs. These findings have initiated a completely new avenue of research by establishing -1 PRF on cellular mRNAs, and its regulation by miRNAs as a new, fundamental paradigm in gene expression. This proposal seeks to deepen our understanding of the molecular mechanisms underlying regulation of -1 PRF in human cells. The well-defined Jurkat human T-cell line and a focus on -1 PRF signals embedded in mRNAs encoding cytokine receptors and a critical cytokine-responsive tyrosine kinase provides a model system to address a series of questions ranging from basic molecular and structural biology to regulation and control of the acquired immune response.
Aim 1 of this proposal seeks to confirm -1 PRF promoted by sequences identified in the mRNAs encoding IL2R?, IL7R?, and JAK2, characterize the effects of SNPs on -1 PRF, and develop the next generation PRF technology.
Aim 2 will identify and validate miRNAs that naturally interact with these -1 PRF signals, and will test an autoregulatory feedback loop model of -1 PRF.
Aim 3 is oriented towards characterizing the effects of miRNAs on gene expression and RNA structure. By the end of the proposed studies, we will have 1) deepened our understanding of this new paradigm gene expression control, 2) identified specific miRNAs that are used by T-cells to control their responses to important cytokines, and 3) established new rules describing mRNA/miRNA atomic scale structural interactions. These studies will immediately impact many fields including basic molecular and cell biology, and more applied fields including immunology and HIV/AIDS, and will lay the foundation for the design and discovery of small molecule therapeutics targeted to specific -1 PRF signals.
Programmed -1 ribosomal frameshifting (-1 PRF) is a fundamental molecular mechanism that is used to control gene expression from yeast to humans by destabilizing messenger RNAs. Building on our recent demonstration of sequence-specific regulation of -1 PRF by micro-RNAs (miRNAs), we will identify the miRNAs that are naturally used by cells to regulate -1 PRF in a subset of mRNAs involved in cytokine responses of human T-cells, and will characterize the relationship between mRNA/miRNA structures on -1 PRF and gene expression. The studies proposed in this application will expand our understanding of RNA structure/function relationships and lay the foundation for the design of small molecule therapeutics targeted to specific -1 PRF signals.
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