The Role of mRNA 3’ End Processing in Eukaryotic Gene Expression
The goal of our research is to understand the molecular mechanism of mRNA 3’ end formation and how it contributes to the appropriate expression of eukaryotic genes. In this essential phase of mRNA biogenesis, primary transcripts are cleaved and then further processed by the addition of a tract of adenosine residues. The execution of this processing step prevents readthrough transcription from interfering with DNA elements such as promoters, centromeres, and replication origins. In addition, acquisition of the poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its timely removal when the mRNA is no longer needed by the cell. Maturation of mRNA 3’ ends also serves as an important point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Furthermore, it has been linked to other essential processes such as chromosome segregation, DNA repair, and tissue-specific protein expression. Mistakes in polyadenylation can impact on all of these processes.
Our lab uses the complementary approaches of biochemistry, genetics and molecular biology to study mRNA 3’ end processing in the model eukaryote, the yeast Saccharomyces cerevisiae, Some of the on-going projects in the lab are described below:
The Molecular Mechanism of mRNA 3’ End Formation
The cleavage and polyadenylation of mRNA occurs in a surprisingly large complex that is well conserved from yeast to mammals (Figure 1). In yeast, this complex has 21 proteins! However, little is known about how all of these proteins cooperate with each other to insure processing that is accurate and efficient. We hypothesize that rearrangements of protein partners occur within the complex as it evaluates the authenticity of the processing site, commits to cleavage at the poly(A) site, reorganizes to position the poly(A) polymerase for tail synthesis, and releases the final RNA product once the tail reaches the appropriate length. We want to understand how such reorganizations drive the cycle of mRNA 3’ end processing, and how these transitions are regulated by post-translational modifications and interactions with the transcription and export machineries. We are also collaborating with Dr Andrew Bohm in Biochemistry to define the structure of the processing complex and how this architecture contributes to function.
Figure 1. The yeast mRNA 3’ end processing complex is composed of three factors that can be separated biochemically. CPF contains the endonuclease Ysh1 and the poly(A) polymerase Pap1. The CF IA and Hrp1 factors help position CPF at a poly(A) site.
Coordination with Other Processes Involved in mRNA Synthesis
Another goal of our research is to examine how the polyadenylation machinery is linked with other steps in mRNA synthesis (Figure 2). Coupling processing with synthesis allows the cell to achieve greater efficiency and provides a checkpoint to insure that transcription termination happens only after the 3’ end processing complex is assembled onto the nascent transcript. In turn, the assembly of the processing complex is thought to alter the processivity of RNA Polymerase II, and the subsequent cleavage at the poly(A) site provides an entry site for a 5'-3- exonuclease, leading to transcription termination farther downstream. The assembly of a ribonucleoprotein (RNP) particle that can be efficiently exported from the nucleus is also closely integrated with 3’ end processing, and the accuracy and completion of these steps is carefully monitored by a nuclear surveillance mechanism that destroys improperly formed mRNPs. Several projects in our lab are dissecting the molecular crosstalk involved in the coordination of polyadenylation with transcription, mRNA transport, and quality control.
Figure 2. The coordination of mRNA transcription, processing and export.
Identification of Small Molecule Inhibitors of mRNA 3’ End Formation
One goal of our lab is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. In the last few years, our research and that of others has identified most, if not all, of the subunits of the 3’ end processing complex and revealed a remarkable conservation between the yeast Saccharomyces. cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3’ end formation could be found. We are currently analyzing candidate inhibitors obtained from a high through-put screening of 300,000 small molecules and designing new screens to target different points in the pathway of mRNA 3’ end synthesis.