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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. |