Single-molecule studies of mRNA transport, processing, and localization
Analysis of gene expression is usually performed on messenger RNAs obtained from homogenized tissues. The gene
expression profiles thus obtained represent the ?averaged? behavior of all cells in the tissue and mask variations that
may exist between cells. We have developed probes that permit detection and imaging of individual molecules of
mRNAs within single cells. Using these probes we carried out a ?census? of mRNAs within cells found that individual
cells of higher eukaryotes display massive cell-to-cell variations in gene expression. These variations arise because
the mRNAs are produced in randomly initiated bursts. This stochastic mRNA synthesis explains why in a population of
genetically identical stem cells only some cells would proceed to differentiate; and why a fraction of cells within tumors
display resistance to drugs.----We used the same approach to study the intracellular venues of splicing, the process
that links the meaningful portions of the genes with each other while eliminating the intervening non-coding
sequences. Usually the splicing occurs at the gene locus, while the pre-mRNA is being synthesized. However, with our
single-molecule approach we found that during regulated alternative splicing, the normally tight coupling of
transcription and splicing is broken, and the pre-mRNAs are spliced after their release from the gene locus in the
nucleoplasm. As the alternative splicing is widespread, and is key to the cell type specification, this discovery will be
important to our understanding how widely different cell types, like neurons and bone cells, are obtained from
otherwise indistinguishable progenitor cells.---- A number of different mRNA species travel to synaptic regions of
dendrites in neurons of hippocampus. At thesynaptic sites these mRNAs produce proteins that are needed in the
synaptic development. Our single-molecule imaging studies in neurons revealed that mRNAs travel as single
molecules rather than as clusters of many molecules to the synaptic regions of the neurons. These observations have
important implications to our understanding of cellular basis of memory formation.----Other investigators are
utilizing our single molecule imaging approach to study diverse biological phenomenon, ranging from packaging of
RNA within influenza virus, stem cell differentiation, biology of model organisms, like worms and fruit flies, to imaging
of RNAs within tumors.
