Michael Rosbash is interested in RNA processing and the
genes and mechanisms that underlie circadian rhythms.
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.)
Rhythms and Behavior
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.
Selected Publications:
A Novel Plasmid-Based Microarray Screen Identifies Suppressors
of rrp6{Delta} in Saccharomyces cerevisiae. Abruzzi
K, Denome S, Olsen JR, Assenholt J, Haaning LL, Jensen TH,
Rosbash M.m Mol Cell Biol. 2007 Feb;27:1044-55.
[abstract]
A genome-wide analysis indicates that yeast pre-mRNA splicing
is predominantly posttranscriptional, Tardiff DF, Lacadie
SA, Rosbash M. Mol Cell. 2006 Dec 28;24:917-29.
[abstract]
3'-end formation signals modulate the association of genes
with the nuclear periphery as well as mRNP dot formation.
Abruzzi KC, Belostotsky DA, Chekanova JA, Dower K, Rosbash
M. EMBO J. 2006 Sep 20;25:4253-62. [abstract]
Neurotoxic protein expression reveals connections between
the circadian clock and mating behavior in Drosophila.
Kadener S, Villella A, Kula E, Palm K, Pyza E, Botas J,
Hall JC, Rosbash M. Proc Natl Acad Sci U S A. 2006
Sep 5;103(36):13537-42. Erratum in: Proc Natl Acad Sci
U S A. 2006 Nov 7;103:17065. [abstract]
In vivo commitment to yeast cotranscriptional splicing
is sensitive to transcription elongation mutants.Lacadie
SA, Tardiff DF, Kadener S, Rosbash M. Genes Dev.
2006 Aug 1;20:2055-66. [abstract]
Arrested yeast splicing complexes indicate stepwise snRNP
recruitment during in vivo spliceosome assembly.Tardiff
DF, Rosbash M. RNA. 2006 Jun;12:968-79. [abstract]
PDF cycling in the dorsal protocerebrum of the Drosophila
brain is not necessary for circadian clock function. Kula
E, Levitan ES, Pyza E, Rosbash M. J Biol Rhythms.
2006 Apr;21:104-17. [abstract]
A resetting signal between Drosophila pacemakers
synchronizes morning and evening activity. Stoleru D, Peng
Y, Nawathean P, Rosbash M. Nature. 2005 Nov 10;438:238-42.
[abstract]
Cotranscriptional spliceosome assembly dynamics and the
role of U1 snRNA:5'ss base pairing in yeast. Lacadie
SA, Rosbash M. Mol Cell. 2005 Jul 1;19:65-75.
[abstract]
PERIOD1-associated proteins modulate the negative limb
of the mammalian circadian oscillator. Brown SA, Ripperger
J, Kadener S, Fleury-Olela F, Vilbois F, Rosbash M, Schibler
U., Science. 2005 Apr 29;308:693-6. [abstract]
Assaying the Drosophila negative feedback loop with
RNA interference in s2 cells.Nawathean P, Menet JS, Rosbash
M. Methods Enzymol. 2005;393:610-22. [abstract]
Nonsense-mediated decay does not occur within the yeast
nucleus. Kuperwasser N, Brogna S, Dower K, Rosbash M. RNA.
2004 Dec;10:1907-15. [abstract]
A synthetic A tail rescues yeast nuclear accumulation of
a ribozyme-terminated transcript. Dower K, Kuperwasser N,
Merrikh H, Rosbash M. RNA. 2004 Dec;10:1888-99.
[abstract]
Coupled oscillators control morning and evening locomotor
behaviour of Drosophila. Stoleru D, Peng Y, Agosto
J, Rosbash M. Nature. 2004 Oct 14;431:862-8.
[abstract]
Effects of the U1C L13 mutation and temperature regulation
of yeast commitment complex formation. Du H, Tardiff
DF, Moore MJ, Rosbash M. Proc Natl Acad Sci U S A.
2004 Oct 12;101:14841-6. [abstract]
Roles of the two Drosophila CRYPTOCHROME structural
domains in circadian photoreception. Busza, A., Emery-Le,
M., Rosbash, M., and Emery, P. Science, 304:1503-1506
(2004). [abstract]
Biochemical analysis of TREX complex recruitment to intronless
and intron-containing yeast genes. Abruzzi KC, Lacadie S,
Rosbash M. EMBO J. 23:2620-31.(2004) [abstract]
The doubletime and CKII kinases collaborate to potentiate
Drosophila PER transcriptional repressor activity. Nawathean,
P. and Rosbash, M. Mol Cell 13:213-223 (2004)
[abstract].
The Co-evolution of Blue-Light Photoreception and Circadian
Rhythms. Ghering, W. and Rosbash, M. J. Mol. Evol,
57:S286-S289 (2003). [abstract]
Drosophila free-running rhythms require intercellular communication.
Peng, Y., Stoleru, D., Levine, J.D., Hall, J.C. and Rosbash,
M. PLOS 1:32-40 (2003). [abstract]
Localization of nuclear retained mRNAs in Saccharomyces
cerevisiae.. Thomsen, R., Libri, D., Boulay, J., Rosbash,
M., and Heick Jensen, T. RNA 9:1049-1057 (2003).
[abstract]
A recessive mutant of Drosophila Clock reveals a
role in circadian rhythm amplitude. Allada, R., Kadener,
S., Nandakumar, N., and Rosbash, M. EMBO J. 22:3367-3375
(2003). [abstract]
Drosophila Clock can generate ectopic circadian
clocks. Zhao, J., Kilman, V., Keegan, K., Peng, Y., Emery,
P., Rosbash, M., and Allada, R. Cell 113:755-766
(2003). [abstract]
Early formation of mRNP: License for export or quality
control. Heick Jensen, T. Dower, K., Libri, D., and Rosbash,
M. Mol. Cell 11:1129-1138 (2003). [abstract]
A biological clock. Rosbash, M. Daedalus 132: 27-36
(2003).
Co-transcriptional monitoring of mRNP formation. Heick
Jensen, T., and Rosbash, M. Nature Structural Biology 10:10-12
(2003).
Circadian rhythms: The cancer connection. Rosbash, M. and
Takahashi, J.S. Nature 420: 373-374 (2002).
A role for casein kinase 2± in the Drosophila
circadian clock. Lin, J.M., Kilman, V.L.,Keegan, K., Paddock
B., Emery-Le, M., Rosbash, M., and Allada R. Nature 420:
816-820 (2002).
Interactions between mRNA export commitment, 3'-end quality
control and nuclear degradation. Libri, D., Dower, K., Boulay,
J., Thomsen, R., Rosbash, M., and Jensen, T.H. Mol. Cell
Biol. 22:8254-8266 (2002).
The U1 snRNP protein U1C recognizes the 5' splice site
in the absence of base pairing. Du, H. and Rosbash, M. Nature
419:86-92 (2002).
Ribosome components are associated with sites of transcription.
Brogna, S., Sato, T.A., and Rosbash, M. Mol. Cell 10:M
93-104 (2002). [abstract]
Sequential nuclear accumulation of the clock proteins period
and timeless in the pacemaker neurons of Drosophila melanogaster.
Shafer, O.T., Rosbash, M., and Truman, J.W. J. Neurosci.
22: 5946-5954 (2002). [abstract]
Regulation of alternative splicing by a transcriptional
enhancer through RNA pol II elongation. Kadener, S., Fededa,
J.P., Rosbash, M., and Kornblihtt, A.R. Proc. Natl. Acad.
Sci. U.S.A 99:8185-8190 (2002). [abstract]
T7 RNA polymerase-directed transcripts are processed in
yeast and link 3' end formation to mRNA nuclear export.
Dower, K. and Rosbash, M. RNA 8 :686-697 (2002). [abstract]
View Complete Publication List on PubMed:
Michael Rosbash
Last update: February 27, 2007. E-mail comments
or questions to the webmaster.
