What We Do

Our lab is interested in the mechanisms and regulation of mRNA metabolism, specifically polyadenylation and mRNA decay.

mRNA turnover projects

Messenger RNA decay is an often over-looked but highly regulated aspect of gene expression that is integral in determining mRNA levels. Recent studies suggest that as much as 50% of the changes in gene expression that occur in response to certain stimuli are at the level of mRNA stability. We have developed and patented an in vitro assay that allows us to recapitulate the various steps of mRNA decay in S100 extracts from multiple cell types including HeLa, Jurkat, and even trypanosomes (Ford et al, 1999) and more recently a mosquito cell line (Opyrchal et al 2005). This assay is integral to almost all of our research efforts and has been utilized to generate results for more than a dozen publications from our lab as well as being adopted by other labs (e.g. Chen et al, Genes Dev 2000; Sengupta et al, J.Biol.Chem. 2004; Tran et al, Mol.Cell. 2004). In eukaryotes, mRNA decay initiates with removal of the poly(A) tail (deadenylation). In our in vitro system, this step is performed by PARN, a cap-dependent deadenylase. We have recently found that an RNA-binding protein, CUG-BP, interacts with PARN and recruits it to certain mRNAs resulting in enhanced deadenylation. Interestingly, CUG-BP expression is disrupted in myotonic dystrophy. Libin Zhang, a post-doc in the lab, is investigating whether the effects of CUG-BP on deadenylation of specific mRNAs may explain some of the pathogenesis seen in myotonic dystrophy. After deadenylation, mRNAs are degraded by one of two pathways, decapping and 5'-3' exonucleolytic decay or 3'-5' exonucleolytic decay. The 3'-5' pathway is mediated by a large complex of exonucleases known as the exosome. John Anderson, along with several former members of the lab, has recently shown that several exosome subunits have specific binding affinity for AU-rich elements (Anderson et al 2006). This is interesting because AU-rich sequences are typically present in the 3' UTRs of unstable mRNAs.

Virus Projects

We have initiated collaborations with the arboviral research groups here at CSU in order to study how viruses are able to evade the host cell mRNA degradation machinery to persist and replicate in the cytoplasm. Mateusz Opyrchal, an MD/PhD student who recently graduated, was able to adapt our in vitro system to mosquito cells and has shown that the Sindbis virus 3'UTR can act as an inhibitor of deadenylation in mosquito extracts. Nicole Garneau, and Kevin Sokoloski have now taken over aspects of this project as graduate students. We are also interested in the late mRNAs encoded by the poxvirus. These mRNAs are unusual in that they have poly(A) tracts of 5-35 residues at their 5' ends. Naomi Bergman, who recently graduated, and Karen Moraes, who has now started her own lab in Brazil, examined the role of the 5' A tract as an inhibitor of mRNA decay in vitro and in cells. This project is being continued by Kevin Sokoloski.

Polyadenylation Projects

Polyadenylation is a co-transcriptional process by which a poly(A) tail is added to the 3' end of mRNAs. The poly(A) tail is both a determinant of mRNA stability and a translation enhancer. These effects are mediated through interaction of poly(A) with the poly(A) binding protein. Although, the machinery and signals for optimal polyadenylation are quite well-characterized, a large number of mRNAs do not contain canonical poly(A) signals. In collaboration with Dr Carol Lutz, and Dr Bin Tian at New Jersey Medical School, Angie Morrison is currently trying to decipher how these non-canonical signals work. Several years ago, it was found that the process of splicing deposits a mark on the mRNA which influences downstream events such as export and translation. We wondered whether polyadenylation marks the mRNA body in a similar fashion. Visu Palaniswamy, a post-doctoral fellow who is now in David Wong's lab in Los Angeles, discovered that nucleophosmin, an abundant protein implicated in cancer, is deposited on mRNAs as a result of polyadenylation. Angie Morrison, a Masters student, and Hend Ibrahim, a post-doc, are currently investigating the requirements for nucleophosmin binding as well as trying to determine the effects of nucleophosmin on downstream steps of mRNA metabolism such as mRNA export, splicing and translation.

Other projects in the lab are too preliminary for such a public forum, but we would be happy to discuss them with interested students and post-docs.