Post-transcriptional controls are critical to the establishment of selective patterns of gene expression. Efforts to identity and characterize the protein complexes that assemble on target mRNAs and control their cytoplasmic stability, localization, and translation are therefore of central importance in understanding pathways of cell differentiation and function. The alpha-globin polyC-binding proteins (alphaCPs) comprise a widely distributed and highly abundant family of RNA binding proteins. alphaCPs were initially identified as critical components of the human alpha-globin mRNA 3'UTR stabilization complex. Subsequent studies have revealed that alphaCPs can selectively bind to a variety of regions within target mRNAs, have a characteristic preference for binding to single-stranded C-rich motifs, and mediate a wide spectrum of post-transcriptional controls. In erythroid cells, alphaCPs are involved in stabilization of alpha- and beta-globin mRNAs and translational control of the highly abundant reticulocyte 15-lipoxygenase mRNA. The pathways associated with the distinct sets of post-transcriptional controls mediated by alphaCP-RNP complexes remain poorly understood. I have recently identified a comprehensive set of cytoplasmic mRNAs targeted by alphaCP2 in a human erythroid-related cell line (K562 cells) (MCB 23;7055 2003).
The aim of the proposed fellowship is to characterize and organize these alphaCP2-mRNA interactions into functional groups with the goal of defining how the sites and structures of this prototype RNP complex can dictate the type of post-transcriptional control that it mediates. In this fellowship I propose three Aims.
In Aim I a high-throughput approach will be utilized to determine the functional impact of alphaCP2 binding on mRNA targets in K562 cells.
In Aim II the position, structure, and sequence of selected alphaCP2 binding sites that correspond to each of the defined alphaCP2-dependent post-transcriptional controls will be determined.
In Aim III informatic approaches will be used to establish models from the data generated by Aims I and II. These models may be able to describe and potentially predict alphaCP2 RNP function. The strength of these models will then be tested by functional analyses of novel alphaCP2 binding sites on mRNAs predicted by our algorithms. The results from this proposal will substantially expand our understanding of how gene expression is regulated in erythroid cells, potentially identify targets for therapeutic interventions, and may serve as a general prototype to predict functional implications of additional categories of sequence specific RNA-binding proteins.
| Waggoner, Shelly A; Johannes, Gregg J; Liebhaber, Stephen A (2009) Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest. J Biol Chem 284:9039-49 |
