tumor cell biology program - Sylvester Comprehensive Cancer Center

sis, and proceeds in a manner that has
many similarities to eukaryotic transcription
initiation.
Dr. Malhotra’s laboratory is studying
the mechanism of sigma54-enhancer
directed transcription initiation using X-
ray crystallography. Crystallization work
on sigma54 and an associated activator
protein is in progress. His team is also
looking at the overall architecture of the
sigma54-core RNAP activated complexes
by 3D electron microscopy, and biochemical
approaches such as protein-protein
footprinting and cross-linking.
Negative strand RNA viruses (involved
in diseases such as measles, rabies
and ebola) encode a multifunctional
RNA dependent RNA polymerase,
which performs both transcriptive and
replicative functions and also displays
mRNA capping and polyadenylation
activities. Crystallographic studies of this
polymerase and associated proteins are
in progress in his laboratory.
Ribosomal Structure
Ribosomes are complex ribonucleoprotein
particles responsible for mRNA directed
protein synthesis in all living cells.
The large size of this macromolecular
assembly (the E. coli ribosome has a MW
of about 2.7 million daltons) makes it
necessary to use both electron microscopy
and X-ray crystallography to understand
its structure. His laboratory is
using 3-D electron-microscopy data
from the laboratory of Dr. Joachim
Frank, a Howard Hughes Research Investigator
at the Wadsworth laboratories
in Albany, New York, to understand the
overall architecture of the ribosomal
RNA in the ribosome. In addition, the
structure of some ribosomal proteins and
protein-RNA complexes by X-ray crystallography
is also being investigated.
Structural Studies of Bacterial
Exoribonuclease
Ribonucleases (RNases) play a central
role in several aspects of cellular RNA
metabolism, including mRNA decay
and maturation and turnover of structural
RNAs. In collaboration with the
laboratory of Dr. Murray Deutscher in
the Department of Biochemistry and
Molecular Biology, crystallographic studies
are underway to decipher the atomic
structure of RNase T from Escherichia
coli.
PUBLICATIONS
Malhotra, A and Frank, J. Predicting
three-dimensional structure of ribosomal
RNA using cryo-electron
microscopy maps of the E. coli ribosome.
Nucleic Acid Research Symposium Series
41: 32, 1999.
Akila Mayeda, Ph.D.
Assistant Professor of Biochemistry
and Molecular Biology
DESCRIPTION OF RESEARCH
Dr. Mayeda’s research focuses on the
molecular mechanisms of constitutive
and alternative pre-mRNA splicing.
By taking advantage of biochemical,
molecular-biological, and immunological
approaches together with splicing
assays in vitro and in vivo, Dr. Mayeda’s
team is attempting to purify and clone
novel splicing factors that are involved
in the general splicing pathways and in
the alternative splicing regulations (Figure).
To study the structure, post-translational
modifications, and function of
splicing factors, recombinant proteins
from E. coli or baculovirus are routinely
prepared. Specific antibodies also are
used to characterize the tissue-specific expression
patterns and the subcellular localization
of the factors.
Dr. Mayeda’s team has advanced the
understanding of pre-mRNA splicing
through the isolation and characterization
of two distinct classes of RNA-binding
proteins, known as SR and hnRNP
A/B family proteins. Interestingly, these
two kinds of protein have antagonistic
effects upon the selection of splice sites
and the use of alternative exons (Figure).
Recently his team has purified and characterized
a novel human splicing factor
RNPS1, which is a typical RNA-binding
protein with a single canonical RNArecognition
motif (RRM) and extensive
serine-rich domain in upstream.
Human RNPS1 was identified as a general
splicing activator but it would be also
a potential alternative splicing regulator.
Further characterizations of
RNPS1 together with its associated factors
are ongoing research projects in his
laboratory.
Exon-Inclusion/Skipping
Mutually Exclusive Exons
Alternative 3’ Splice Sites
Alternative 5’ Splice Sites
Retained Intron
Patterns of constitutive and alternative pre-mRNA splicing.
White and black boxes indicate constitutively spliced exons and alternatively
spliced exons, respectively. Possible splicing patterns are shown.
12
UM/Sylvester Comprehensive Cancer Center Scientific Report 2002