MAJOR RESEARCH INTERESTS

We are interested in the posttranscriptional processing of eukaryotic messenger RNA and its role in the regulation of gene expression. Specifically, our research involves a genetic and biochemical analysis of factors that catalyze cleavage and polyadenylation of messenger RNA precursor in the yeast Saccharomyces cerevisiae. This investigation should lead to an understanding of how these factors interact with each other and the RNA substrate in order to process the RNA and how these activities are regulated during the cell cycle or during different growth conditions. We are also studying how the process is regulated by post-translational modifications such as phosphorylation. Another goal is to examine how the polyadenylation machinery interconnects with other processes involved in mRNA biosynthesis, such as transcription, degradation of improperly processed transcripts and nuclear export.

Accurate processing of the 3'-end of the primary RNA transcript is an essential step in the mRNA maturation of all eukaryotes. The process of polyadenylation is important for transcription termination and the resulting poly(A) tail has been implicated in numerous aspects of RNA metabolism including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. Some of the on-going projects in the lab concerning mRNA 3'-end formation are described below.

1. Analysis of factors which catalyze the cleavage and polyadenylation of mRNA precursor

Mechanistically, polyadenylation consists of a tightly coupled two-step reaction: a site-specific endonucleolytic cleavage of the pre-mRNA, followed by the processive synthesis of a poly(A) tail of defined length onto the 3'-end of the upstream cleavage product. This requires the presence of cis-acting signal sequences in the untranslated region of the pre-mRNA as well as trans-acting protein factors. The ability to uncouple cleavage and poly(A) addition in vitro has allowed the biochemical identification of factors involved in either one or both steps of the process.

In Saccharomyces cerevisiae, cleavage requires cleavage/polyadenylation factor I (CF I) and Cleavage factor II (CF II), while tail synthesis requires poly(A) polymerase (Pap1), cleavage factor I (CF I) polyadenylation factor I (PF I), and Pab1 or Nab2. CF II, Pap1, and PF I can be isolated as a larger complex called CPF (Figure 1). A combination of biochemical and genetic approaches has identified almost all of the genes involved in this process. This work has revealed a striking degree of conservation from yeast to mammals among the proteins components required for polyadenylation, despite substantial differences in the signals on the pre-mRNA. A major focus of our lab is to understand how these factors recognize signals on the RNA and assemble into a functional processing complex, how cleavage is accomplished, how the complex transitions from the cleavage step to the poly(A) addition step, how the activity of the poly(A) polymerase is regulated so that it acts only on cleaved precursor and synthesizes a tail of the correct length, and how factors are released from the RNA once processing is complete.

Figure 1. The mRNA 3’ end processing complex in Saccharomyces cerevisiaerase

2. Analysis of the structure and function of poly(A) polyme

As the catalytic subunit, poly(A) polymerase (PAP) is at the heart of the machinery required for poly(A) addition. We are interested in how the structure of poly(A) polymerase makes it an efficient terminal adenylyltransferease and one with remarkable specificity for ATP and RNA as substrates. Towards this end, we are analyzing the effects of mutations in amino acids predicted to be important for these functions. In collaboration with Dr. Andrew Bohm in the Biochemistry Department at Tufts, we have solved the crystal structure of the yeast PAP (Pap1) and found that the enzyme is organized into three domains of 150-200 amino acids each (Figure 2). The N-terminal domain belongs to the nucleotidyl transferase (NT) superfamily of enzymes, and contains the hallmark triad of carboxylic amino acids. These carboxylic amino acids participate in the metal-coordinated binding of the ATP phosphate groups, and are responsible for positioning the nucleophilic 3'-OH of the primer to facilitate the nucleotidyl transferase reaction. The middle domain of PAP has no overall sequence (or structural) homology to other proteins. Our structural and mutagenesis studies suggest that this region interacts with the phosphates of the incoming ATP and functions in removing the pyrophosphate byproduct of the reaction and in translocation of the RNA in preparation for another round of adenylation. The C-terminal region of PAP is responsible for holding the single stranded poly(A) primer in place. All of the other polymerases solved to date use a "thumb domain" to hold onto their substrate via the template strand. Since PAP does not utilize a template, the thumb domain is missing, and the responsibility for holding the substrate has been largely shifted to the C-terminal domain. Our current research is aimed at determining the structure of Pap1 in complex with both RNA and ATP, at trapping transition states in the reaction, and at examining the complex of Pap1 and the regulatory protein Fip1.

Figure 2. The structure of yeast poly(A) polymerase

3. Coupling of polyadenylation with mRNA transcription and transport

In vitro studies have clearly demonstrated that the reactions of transcription, capping, splicing, and mRNA 3’ end formation can take place independently. However, recent work in both yeast and mammalian cells indicates that in vivo, the cell creates the equivalent of an assembly line that couples all of these processes on each RNA Polymerase II transcript. An interaction of RNAP II and the polyadenylation machinery is important for efficient 3’ end formation. In turn, the assembly of the processing complex is thought to somehow alter the processivity of the RNAP II complex and execution of the cleavage step provides an entry site for a 5’-3- exonuclease, leading to transcription termination farther downstream. Recent studies also suggest that transport factors are deposited onto the RNA during splicing and 3’-end formation in a way that leads to the assembly of a complex which can be efficiently transported. The accuracy and completion of processing is carefully monitored by a nuclear surveillance mechanism that destroys improperly processed mRNA. Several projects in our lab are designed to understand the molecular mechanism involved in the coordination of polyadenylation with transcription, mRNA transport, and quality control.