RNA
PROCESSING
Primary pre-mRNA transcripts undergo numerous processing events in the nucleus.
The resulting mature mRNA is then exported to the cytoplasm. Nuclear processing
includes capping, splicing, and polyadenylation, which are the major covalent
modifications experienced by pre-mRNA. These changes depend on the noncovalent
recruitment of factors to RNA, i.e., on the formation of RNA-protein (RNP) complexes.
Nuclear RNP formation can also contribute to protein synthesis, because nuclear
RNA-binding proteins can enhance RNA export from the nucleus and in some cases
remain associated with the mRNA to increase translation efficiency in the cytoplasm.
In addition, nuclear pre-mRNA-processing steps are intimately connected with
transcription. This is due in part to the cotranscriptional recruitment of RNA-binding
proteins and other factors to nascent RNA, by the transcriptional machinery
as well as by the nascent RNA itself. Cotranscriptional protein recruitment
to nascent RNA may increase the efficiency of proper mRNP (messenger RNA–protein)
formation. Another possibility is that it allows the early monitoring of mRNP
quality, so that "bad" mRNP can be prevented from reaching the cytoplasm.
Indeed, the failure to form a proper mRNP apparently leads to mRNA retention
by a transcription site–associated surveillance system and ultimately
to nuclear mRNA degradation.
We are interested in several aspects of these cotranscriptional mRNP assembly
and surveillance issues, and we study them primarily in the yeast Saccharomyces
cerevisiae because of its genetic advantages. Yeast pre-mRNA splicing is an
object of attention, as there is good evidence that splice site identification
and splice site partner assignment rely on the earliest interactions between
the pre-mRNA substrate and a subset of splicing factors. These include the recruitment
of U1 snRNP (U1 small nuclear RNP), which commits pre-mRNA to the splicing pathway
by forming a "commitment complex" with pre-mRNA during in vitro splicing.
Indeed, yeast U1 snRNP recruitment occurs co-transcriptionally in vivo, essentially
coincident with transcription of the intronic RNA. Recruitment of the rest of
the splicing snRNPs and splicing itself then occurs subsequently. Although these
later events can also occur cotranscriptionally, our current view is that they
occur predominantly post-transcriptionally. This is because second exons of
most yeast intron-containing genes are short, so polyadenylation generally precedes
most splicing factor recruitment and splicing.
The recruitment of splicing factors and other mRNA-binding proteins to nascent
mRNA almost certainly involves chromatin as well as RNA polymerase II and other
elements of the transcriptional machinery. Indeed, many factors are probably
prerecruited by DNA bound proteins and then transferred during transcription
to nascent RNA. Our interest in chromatin and the transcription machinery has
been heightened by three recent findings. First, we have done a genetic screen
that implicates transcription machinery components in splicing efficiency. Second,
we have been working on experiments that implicate mRNP and chromatin components
in tethering active genes to the nuclear periphery. Third, we have been able
to purify active chromatin containing RNA polymerase II; RNP proteins are copurified,
indicating an association of these components to elongating polymerase. (A grant
from the National Institutes of Health provided support for the RNA and yeast
projects.)
CIRCADIAN RHYTHMS
When we began our studies on the circadian rhythms of Drosophila about
25 years ago, our goal was to define the machinery that underlies the almost
ubiquitous process of circadian rhythmicity. Our entrée into this problem
was the period gene (per) of Drosophila melanogaster, discovered more than 10
years earlier in pioneering behavioral genetic experiments by Ronald Konopka
and Seymour Benzer. My hope was that defining the per protein (PER) and its
function would lead to some understanding of the biochemical mechanism of circadian
timekeeping. In 1990, we discovered that per mRNA as well as PER undergoes fluctuations
in level during the circadian cycle. These observations and others showed that
there is a negative-feedback loop, in which PER inhibits the transcription of
its own mRNA. Temporally controlled negative feedback at the transcriptional
level is now an accepted feature of circadian timekeeping in plants, cyanobacteria,
Neurospora, and even mammals. Moreover, the approximately 12 additional clock
components defined by Drosophila genetics over the past decade are largely conserved
with mammals and perform similar functions in the mammalian clock, indicating
that the machinery as well as the principles of the Drosophila clock is widely
conserved.
Although we are still interested in defining new clock components, we now have
two more pressing goals: (1) to understand in more biochemical detail how the
Drosophila circadian clock works—for example, what keeps 24 hour time
and what is the relationship of the endogenous pacemaker to the external light-dark
cycle? (2) to define and understand the neural circuit in the fruit fly brain
that underlies the circadian rest-activity cycle.
In pursuit of the first goal, my lab is working on the regulation of clock protein
function and its relationship to CRY, the major Drosophila photoreceptor protein.
(CRY is the product of the cryptochrome gene.) Surprisingly, PER and its partner
protein TIM (the timeless or tim protein) interact with CRY not only in response
to phase-shifting light pulses but also to robust heat pulses. It is remarkable
that a photoreceptor is involved in temperature phenomena, and there is evidence
that the failure to respond to sub-optimal heat pulses is related to critical
temperature compensation (temperature-insensitivity) features of the circadian
clock. We are also interested in the relationship of the CRY interaction to
post-transcriptional as well as transcriptional regulation of clock proteins.
With respect to transcriptional regulation, we continue to study the two key
circadian transcription factors, Clock (CLK) and Cycle (CYC). This key heterodimeric
complex drives per and tim transcription as well as the transcription of other
important genes in a circadian manner. We are interested in the mechanism of
temporal repression, and we are using a variety of strategies to identify additional
circadian genes that contribute to transcriptional regulation.
In pursuit of the second goal, we are focusing on various brain-anatomical aspects
of Drosophila rhythms. There are only six neuronal groups, comprising about
75 pairs of cells, which express high levels of clock genes in the adult brain.
One group controls the characteristic morning activity peak of the insect activity
pattern, and another controls the evening peak. In addition, the morning oscillator
is the master pacemaker under constant darkness conditions and sends a daily
resetting signal to the evening oscillator, which ensures that the two groups
stay in sync. Recent experiments show that the relationship between the two
oscillators switches as a function of environmental conditions, and the evening
cells are the masters in constant light. Moreover, the data indicate that this
inter-oscillator communication serves as a seasonal timer, to adjust locomotor
activity rhythms to day length changes. We are trying to develop assays that
will allow us to visualize these clock neurons and circuits in real time as
well as under different environmental conditions. (Grants from the National
Institutes of Health provided support for the biochemical and genetic approaches
to identify additional clock components and to study neuronal circuits.)
Last updated: February 26, 2007