Research Interests
Our lab employs a biochemical approach to study post-transcriptional regulatory mechanisms in eukaryotic gene expression. More specifically, we are interested in characterizing the interplay between proteins involved in RNA decay pathways. The life span of an individual mRNA represents a post-transcriptional opportunity for cells to influence the synthesis of nascent peptides. We focus on two main objectives: (i) to identify the determinants and define their regulatory roles in eukaryotic RNA stability and (ii) to define the regulatory roles of RNA-binding proteins in specific human genetic disorders.
(I a) Eukaryotic RNA Stability -Regulatory mechanisms of mRNA decay pathway
As a determinant of mRNA half-life, the physical interaction between trans factors (ie. RNA-binding proteins) and cis elements influences the cellular level of gene product. To identify trans factors which contribute to gene-specific mRNA stability, we utilize the unusually stable a-globin mRNA as a model system in an in vitro mRNA decay assay that recapitulates the physiologically regulated mRNA stability in vivo (Wang, Z., et al., 1999). Stability of the a-globin mRNA is mediated by a specific mRNP complex (a-complex) which 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 mRNA deadenylation, the rate-limiting step in mRNA turnover (Wang, Z., et al., 1999). Second, it protects the mRNA from cleavage by an erythroid-enriched endoribonuclease (ErEN) (Wang, Z. and Kiledjian, M. 2000). Third, it impedes the 3' to 5' exoribonucleolytic decay activity of the exosome (Rodgers, N. D., et al. 2002).
We are also interested in general mammalian mRNA decay, particularly the identification and characterization of the nucleases involved. Two major exoribonuclease-mediated decay pathways have been identified in which mRNA can be cleared from either the 5' or 3' end following removal of poly(A) tail. We have demonstrated a prominent role for both pathways in mammalian mRNA decay and identified a potent 3?to 5?exoribonuclease-dependent scavenger decapping activity (DcpS) that functions following mRNA decay from the 3?end of the mRNA to hydrolyze the residual cap (Wang,
Z. and Kiledjian, M. 2001). DcpS was found to exclusively hydrolyze capped dinucleotides or capped oligonucleotides. Its purification and cloning of the gene encoding it revealed it is a member of the Histidine Triad (HIT) family of hydrolases (Liu, H., et al. 2002). Structural analysis revealed that DcpS is a dynamic structure whereby an N-terminal domain of the protein can ratchet back and forth to alternatively create a decapping competent active site to hydrolyze the cap structure (Gu, M., et al. 2004). Interestingly, we recently demonstrated that the decapping activity of the yeast homolog of DcpS is also involved in a positive feedback mechanism to enhance 5?to 3?exonuclease activity (Liu, H. and Kiledjian, M. 2005). We are currently exploring the mechanism by which this regulation occurs.
A second mRNA decapping enzyme in mammals hDcp2 was also identified and cloned in our lab termed hDcp2. hDcp2 an essential component in the 5' decay pathway and specifically removes the cap moiety from capped RNA but not capped oligonucleotides (Wang, Z., et al. 2002). Its dependence on capped RNA is based on the fact that it is an RNA-binding protein that recognizes the cap only within the context of an RNA (Piccirillo, C., et al. 2003). The RNA-binding property of hDcp2 indicates that this protein will also preferentially associate and regulate a subset of high affinity target mRNA. Efforts are underway to isolate these hDcp2 substrates. We have therefore identified and are analyzing the two decapping nucleases involved in the major mammalian mRNA exonuclease decay pathways.
Our efforts with these projects are currently focused on identification of proteins and compounds that regulate both decapping enzymes and in turn control mRNA stability and gene expression.
(I b) Eukaryotic RNA Stability -Mechanism and regulation of microRNA turnover
MicroRNAs (miRNAs) are endogenous ~22 nt RNAs that can play important regulatory roles in all metazoan eukaryotes by targeting mRNAs for degradation or translational repression. Whereas the biogenesis and processing of miRNAs have been extensively investigated, the degradation of these noncoding RNAs remains an elusive area of research. miRNAs are often reported to be present and functional during certain early developmental stages, and their cellular levels drop abruptly at later stages. Although these studies suggest that miRNAs are biologically and functionally relevant molecules, the precise manner by which miRNAs are cleared immediately in a developmental or general context has not been mechanistically addressed. We have initiated the study of miRNA turnover and seek to determine whether differential miRNA stability contributes to their abundance and determine the regulatory components control their stability and cellular levels.
(II) Role of RNA-Binding Proteins in Human Disorders
We have devised a strategy termed isolation of Specific Nucleic Acids Associated with Proteins (SNAAP) that enables rapid and efficient isolation of specific cellular mRNA bound by an RNA-binding protein (RNP) (Trifillis, P., et al. 1999;
Carr-Schmid, A., et al. 2006). In particular, we are interested in identifying substrate mRNAs that are specifically bound by distinct RNPs involved in human genetic disorders. Identification of the cognate target mRNA will improve our understanding of the role that these proteins play in certain 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, X., et al. 2002). Our current efforts are focused on characterizing the interaction of DAZL protein with its mRNA partners and understanding how this interaction regulates mRNA target expression and affects spermatogenesis.
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