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RNA-Protein interactions in the regulation of mammalian mRNA turnover. RNA-binding proteins in human genetics disordersThe lab is interested in post-transcriptional regulation of eukaryotic gene expression. in particular. the role of RNA-binding proteins and nucleases that influence the fate of mRNA. There are two main research focuses in the lab. 1) to understand the determinants that regulate mammalian mRNA stability and 2) to characterize the mechanistic role of various RNA-binding proteins in specific human genetic disorders. I. Mammalian mRNA Turnover All phases of mRNA production are precisely regulated and each mRNA has an intrinsic half-life. which influences expression of its gene product. Trans factors (RNA-binding proteins) interact with distinct cis elements to impact mRNA turnover. We are using the unusually stable a-globin mRNA as a model system to study the components of mRNA stability. The globin mRNAs are among the most stable eukaryotic transcripts and provide an ideal system to study the mechanism by which mRNA can be stabilized. We have devised an in vitro mRNA decay assay that recapitulates regulated mRNA stability observed in cells (Wang et al.. 1999). This system has enabled us to identify the trans factors that contribute to the stabilization of mRNA and provided a convenient experimental system to characterize the mechanism by which they function. Stability of the a-globin mRNA is mediated by a specific mRNP complex that forms on the 3' untranslated region (3'UTR). This complex stabilizes the mRNA by at least three mechanisms. First. it increases the binding affinity of the poly(A)-binding protein to the poly(A) tail and prevents deadenylation of the mRNA (Wang and Kiledjian. 2000b). Second. it protects the mRNA from cleavage by an erythroid-enriched endoribonuclease (ErEN; Wang and Kiledjian. 2000a; Rodgers et al.. 2002). Third. it impedes the 3' to 5' exoribonucleolytic decay activity of the exosome (Rodgers et al.. 2002). We are also analyzing the general mechanism of mammalian mRNA decay and the identification and characterization of the nucleases involved. We have recently demonstrated that following deadenylation of mRNA in mammals. the mRNA primarily degrades from the 3' end both in vitro and in cells (Wang and Kiledjian. 2001; Rodgers et al.. 2003). An exoribonuclease-dependent scavenger decapping activity (DcpS) was identified that functions following decay of the mRNA and hydrolyzes the residual cap. The decapping activity can be detected associated with a subset of 3' to 5' exonucleases. implying a higher order degradation complex could coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA. degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity (Wang and Kiledjian. 2001). We have purified and cloned the gene encoding DcpS which is a member of the HIT family of hydrolases (Liu et al.. 2002) and are in the process of characterizing the DcpS protein and activity. We have also cloned a gene encoding a second mRNA decapping activity in mammals. hDcp2 (Wang et al.. 2002). hDcp2 is an RNA binding protein that recognizes and hydrolyzes the cap by a prior association with the RNA (Piccirillo et al. 2003). hDcp2 decapping activity is negatively regulated at multiple levels and we are currently determining how hDcp2 contributes to mRNA decay and how it is regulated. Our efforts with these projects are currently focused on three aspects: A) purification and cloning of the sequence specific endoribonuclease ErEN. B) determining the general pathway of mammalian mRNA turnover and the components involved and C) identify the regulators and the mRNA decay nucleases. II. Role of RNA-Binding Proteins in Mental Retardation and Azoospermia. We have devised a strategy termed isolation of Specific Nucleic Acids Associated with Proteins (SNAAP; Trifillis et al.. 1999) that enables rapid and efficient isolation of specific cellular mRNA bound by an RNA-binding protein. In particular we are interested in identifying substrate mRNAs that are specifically bound by distinct RNA-binding proteins involved in human genetic disorders. Identification of the cognate target mRNA will enable a mechanistic understanding of the role these proteins play in the disorders. We are studying a protein termed Deleted in Azoospermia like (DAZL). which is involved in sperm production. The DAZL protein is primarily cytoplasmic and thought to manifest its function through unknown target mRNAs. We have identified testis mRNAs that are specifically bound by the DAZL protein (Jiao et al. 2002). Our current efforts are focused on understanding the binding of the DAZL protein regulates the expression of the target mRNA and how this regulation could affect spermatogenesis. Selected PublicationsLiu H, Kiledjian M. (2007) An erythroid-enriched endoribonuclease (ErEN) involved in alpha-globin mRNA turnover. Protein Pept Lett. 14(2):131-6. Shen V, Kiledjian M. (2006) A view to a kill: structure of the RNA exosome. Cell. 127(6):1093-5. Jiao X, Wang Z, Kiledjian M. (2006) Identification of an mRNA-decapping regulator implicated in X-linked mental retardation. Mol Cell. 24(5):713-22. Shen V. Kiledjian M. (2006) Decapper comes into focus. Structure. 14(2):171-2. Bail S. Kiledjian M. (2006) More than 1 + 2 in mRNA decapping. Nat Struct Mol Biol. 13(1):7-9. Carr-Schmid A. Jiao X. Kiledjian M. (2006) Identification of mRNA bound to RNA binding proteins by differential display. Methods Mol Biol. 317:299-314. Cohen. L.S.. Mihkli. C.. Jiao. X.. Kiledjian. M.. Kunkel. G. and Davis. R.E. (2005). Dcp2 Decaps m2,2,7GpppN - Capped RNAs and its Activity is Sequence and Context Dependent. Mol. Cell. Biol. (in press) Khanna. R. and Kiledjian. M. (2004). Poly(A) binding protein mediated regulation of hDcp2 decapping. EMBO J. 23:1968-1976. Liu. S.W.. Liu. H.. Jiao. X.. Gu. M.. Lima. C.D.. and Kiledjian. M. (2004). Functional analysis of mRNA scavenger decapping enzymes RNA 10:1412-1422. Gu. M.. Fabrega. C.. Liu. S.W.. Liu. H.. Kiledjian. M.. and Lima. C.D. (2004). Insights into the structure. mechanism and regulation of the scavenger decapping activity. Mol. Cell 14:67-80. Piccirillo. C.. Khanna. R. and Kiledjian. M. (2003). Functional Characterization of the Mammalian mRNA Decapping Enzyme hDcp2. RNA 9:1138-1147. Wang. Z.. Carr-Schmid. A. Jiao. X.. and Kiledjian. M. (2002). Dcp2 Protein is a Regulated Mammalian mRNA Decapping Enzyme. Proc. Natl Acad. Sci. USA. 99:12663-12668. Jiao. X.. Trifillis. P. and Kiledjian. M. (2002). Identification of target substrates for the Deleted in Azoospermia-Like Protein. Bio. Reproduction. 66:475-485 Liu. H.. Rodgers. N.. Jiao. X. and Kiledjian. M. (2002). The Scavenger mRNA Decapping Enzyme. DcpS. is a member of the HIT Family of Pyrophosphatases. EMBO J. 21:4699-4708. Rodgers. N.D.. Wang. Z.. and Kiledjian. M. (2002). Regulated -globin mRNA decay is a cytoplasmic event proceeding through a 3' to 5' exosome-dependent decapping. RNA 12:1526-1537. Wang. Z. and Kiledjian. M. (2001). Functional Link between the Mammalian Exosome and mRNA Decapping. Cell. 107:751-762. |
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