Research Interests:Pre-mRNA splicing in yeast
Background
Research
Ongoing projects
Saccharomyces cerevisiae
Background
Within the fields of cell and molecular
biology, my research focuses on gene expression, which encompasses a series
of processes by which the information in a gene is converted into the working
machinery of the cell. Through the study of gene expression, we gain a
better understanding of mechanisms by which cellular differentiation takes
place and the causes of abnormal cell growth and development, which in
humans can result in cancer and other diseases. Summarized by the fundamental
paradigm of molecular biology, which states that DNA makes RNA makes protein,
gene expression involves two basic actions: transcription and translation.
Transcription takes place when the
DNA template, or gene, is synthesized into RNA. The messenger RNA produced
in the process is then translated into protein. In eukaryotic cells, once
the initial RNA, called the primary transcript or pre-messenger RNA, has
been synthesized within the cell’s nucleus, it is modified by RNA processing
and becomes the mature messenger RNA ready to exit the nucleus for translation
into protein, which takes place in the cytoplasm. Most eukaryotic genes
contain three types of sequences: regulatory signals, sequences that code
for proteins, and non-coding sequences that interrupt the coding sequences
but do not have a known function. The synthesis of a primary RNA transcript
yields a pre-messenger RNA that intersperses important information about
how to make the protein along with non-coding sequences. In the nucleus,
the non-informational sequences, called introns, are removed from the pre-messenger
RNA by an RNA processing step called splicing. During splicing the introns
are removed and the informational sequences, called exons for expressed
sequences, are joined together to make the mature messenger RNA ready for
transport to the cytoplasm.
While existence of both informational
and non-informational sequences in eukaryotic genes was identified over
twenty years ago, the precise mechanism by which introns are removed from
pre-messenger RNAs is still under study. Mutations that cause errors in
splicing are often found in genetic diseases and in the development of
cancer. For instance, the disease spinal muscular atrophy, the leading
hereditary cause of infant mortality results from mutations in a gene whose
product is required in the splicing pathway. Therefore, understanding the
details of the splicing process will give important insights into the mechanisms
of spinal muscular atrophy and other genetic diseases.
Research
in my lab
I have been using the budding yeast
Saccharomyces
cerevisiae as a system to study the mechanism of pre-messenger RNA
splicing. S. cerevisiae has many advantages as a model system: it
is a relatively simple eukaryote, its entire genome, over 6,000 genes,
has been sequenced, and the mechanism of pre-messenger RNA splicing has
been evolutionarily conserved between yeast and more complex eukaryotes
such as humans. In addition, budding yeast, unlike more complex eukaryotes,
are easy to grow and store and are amenable to both genetic and biochemical
studies.
Pre-messenger RNA splicing occurs in
a large dynamic complex composed of RNA and protein called the spliceosome.
Two transesterification reactions take place in the spliceosome, which
ultimately serve to remove the intron sequences and bring together the
exons. Factors required for splicing in yeast were initially identified
genetically over thirty years ago by examination of the RNA composition
of mutants that could not grow at high temperatures. Mutants in the splicing
pathway accumulate pre-messenger RNA if grown at high temperatures and
are called PRP mutants, for defective in pre-RNA processing.
>From these and other studies, two groups of factors required for splicing
were identified: spliceosomal factors associated with the small nuclear
RNAs in the spliceosome, and factors extrinsic to the spliceosome.
In Dr. Ren-Jang Lin’s lab at the
City
of Hope I studied an extrinsic splicing factor called Prp2, which is
a protein required prior to the first transesterification reaction in pre-messenger
RNA splicing. Prp2 belongs to a family of proteins called "DEAD-box" proteins,
members of which hydrolyze adenosine triphosphate (ATP) as an energy source,
often using the energy to unwind RNA. The designation "DEAD" refers to
an amino acid motif found in the protein, in which the letters D-E-A-D
are the single letter amino acid code for aspartic acid (D), glutamic acid
(E), and alanine (A). A series of RNA rearrangements occurs in the splicing
pathway; members of the DEAD-box protein family, including Prp2 and the
related DEAD-box proteins Prp16, Prp22, and Prp43, may catalyze these rearrangements.
For more information read a thorough review
of DEAD-box proteins in yeast.
Previous work in Dr. Lin’s lab demonstrated
that the Prp2 protein binds to the spliceosome in the absence of ATP and
is released from the spliceosome following ATP hydrolysis, and in doing
so activates the spliceosome for the first catalytic step of splicing.
We have analyzed the functions of different domains of the Prp2 protein
to determine what regions are required for spliceosome binding and release
as well as ATP hydrolysis activity. We have identified specific amino acids
within the three different domains of Prp2 that are required for these
activities. Both the binding and release regions of Prp2 were identified
using a genetic approach. We constructed mutants in Prp2 that bind to but
do not release from the spliceosome. When a lot of the mutant protein is
made, it interferes with the activity of the normal Prp2 protein in the
cell. If we make a second change in the mutant Prp2 protein that makes
it unable to bind tightly to the spliceosome, it can no longer interfere
with the normal protein. Follow this link to view publications
related to this work.
Ongoing
projects in the lab
1. Examine the role of the C-terminus
of DEAH family members in protein function, specifically spliceosome binding.
2. Explore interactions between
the N- and C-termini of DEAH family members.
3. Uncover the mechanism(s) that
explain the phenotypes of certain alleles of PRP2.
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