rosbashMichael Rosbash, Ph.D.
Professor of Biology
Investigator, Howard Hughes Medical Institute
Member, US National Academy of Sciences

Molecular Genetics of RNA Processing and Behavior

Ph.D., Massachusetts Institute of Technology

contact information
Rosbash lab website

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:

Surprising gene expression patterns within and between PDF-containing circadian neurons in Drosophila. Kula-Eversole E, Nagoshi E, Shang Y, Rodriguez J, Allada R, Rosbash M. Proc Natl Acad Sci U S A. 2010 Jul 12. [abstract]

Light-mediated TIM degradation within Drosophila pacemaker neurons (s-LNvs) is neither necessary nor sufficient for delay zone phase shifts. Tang CH, Hinteregger E, Shang Y, Rosbash M. Neuron. 2010 May 13;66(3):378-85. [abstract]

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA. Menet JS, Abruzzi KC, Desrochers J, Rodriguez J, Rosbash M. Genes Dev. 2010 Feb 15;24(4):358-67. [abstract]

A constant light-genetic screen identifies KISMET as a regulator of circadian photoresponses. Dubruille R, Murad A, Rosbash M, Emery P. PLoS Genet. 2009 Dec;5(12):e1000787. [abstract] [free PMC article]

Dissecting differential gene expression within the circadian neuronal circuit of Drosophila. Nagoshi E, Sugino K, Kula E, Okazaki E, Tachibana T, Nelson S, Rosbash M. Nat Neurosci. 2010 Jan;13(1):60-8. Epub 2009 Dec 6. [abstract]

A role for microRNAs in the Drosophila circadian clock. Kadener S, Menet JS, Sugino K, Horwich MD, Weissbein U, Nawathean P, Vagin VV, Zamore PD, Nelson SB, Rosbash M. Genes Dev. 2009 Sep 15;23(18):2179-91. [abstract] [free PMC article]

A Targeted Bypass Screen Identifies Ynl187p, Prp42p, Snu71p, and Cbp80p for Stable U1 snRNP/Pre-mRNA Interaction. Hage, R., Tung, L., Du, H., Stands, L., Rosbash, M., Chang, TH. MCB 29(14):3941-52 (2009). [abstract]

Genome-wide identification of targets of the drosha/pasha DGCR8 complex. Kadener, S., Rodriguez, J., Abruzzi, K.C., Khodor, Y., Sugino, K., Marr, M., Nelson, S., Rosbash, M. RNA 15:537-545 (2009). [abstract]

The Implications of Multiple Circadian Clock Origins. Rosbash, M. PLoS Biology, 7(3):e62 (2009). [abstract]

Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drsophila brain. Shang, Y., Griffith, L.C., Rosbash, M. Proc.Natl.Acad.Sci. U.S.A. 105:19587-19594 (2008). [abstract]

PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit. Parisky, K.M., Agosto, J., Pulver, S.R., Shang, Y., Kuklin, E., Hodge, J.J., Kang, K., Liu, X., Garrity, P., Rosbash, M., Griffith, L.C. Neuron 60: 672-682 (2008). [abstract]

The nuclear exosome and adenylation regulate post-transcriptional tethering of yeast GAL genes to the nuclear periphery. Vodala, S., Abruzzi, K.C., Rosbash, M. Mol Cell, 31:104-113 (2008). [abstract]

Circadian transcription contributes to core period determination in Drosophila. Kadener, S., Schoer, R., Menet, J.S., Rosbash, M.  PLoS Biology (2008) May 20;6(5):e119. [abstract]

Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila. Agosto, J., Choi, J.C., Parisky, K.M., Stilwell, G., Rosbash, M., and Griffith, L.C.  Nat. Neurosci. 11:354-359 (2008). [abstract]

Sleep: hitting the reset button. Griffith, L.C. and Rosbash, M. Nat. Neurosci. 11:123-124 (2008). [abstract]

Sus1, Sac3 and Thp1 mediate posttranscriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP.  Chekanova, J.A., Abruzzi, K.C., Rosbash, M., and Belostosky, D.A.  RNA 14:66-77 (2008). [abstract]

Transcriptional feedback and definition of the circadian pacemaker in Drosophila and Animals. Rosbash, M., Bradley, S., Kadener, S., Li, Y., Luo, W., Menet, J.S., Nagoshi, E., Palm, K., Schoer, R., Shang, Y., and Tang, C-H. Cold Spring Harb. Symp. Quant. Biol., 72:75-83 (2007).

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking.  Tardiff, D.F., Abruzzi, K.A., and Rosbash, M.  Proc.Natl.Acad.Sci.U.S.A 104:19948-19953 (2007). [abstract]

Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component.  Kadener, S., Stoleru, D., McDonald, M., Nawathean, P., and Rosbash, M. Genes Dev 21:1675-1686 (2007). [abstract]

A small conserved domain of Drosophila PERIOD is important for circadian phosphorylation, nuclear localization and transcriptional repressor activity.  Nawathean, P., Stoleru, D., and Rosbash, M. MCB 27:5002-5013 (2007). [abstract]

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila.  Kaushik, R., Nawathean, P., Busza, A., Murad, A., Emery, P., and Rosbash, M.  PLoS Biology 5:1257-1266 (2007). [abstract]

The Drosophila circadian network is a seasonal timer.  Stoleru, D., Nawathean, P.,  Fernandez, M.P., Menet, J.S., Ceriani, M.F., and Rosbash, M.  Cell 129:207-219 (2007). [abstract]

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].

 

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Last update: July 23, 2010.

 

 
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