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Molecular Genetics of RNA Processing and Behavior
Summary: Michael Rosbash is interested in the regulation of gene expression as well as 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 influence translation efficiency in the cytoplasm.
In addition, nuclear pre-mRNA processing is 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.
We are interested in several aspects of these cotranscriptional mRNP assembly and surveillance issues, and we have studied them primarily in the yeast Saccharomyces cerevisiae because of its genetic advantages. Pre-mRNA splicing has been an object of our attention, and snRNP (small nuclear RNP) recruitment occurs cotranscriptionally in vivo, either coincident with or shortly after transcription of the intronic RNA. Recruitment of the first snRNP, U1 snRNP, is always cotranscriptional; recruitment of the rest of the splicing snRNPs and splicing itself occur subsequently—often post-transcriptionally—in the yeast system. This is because second exons of most yeast intron-containing genes are short, so polyadenylation generally precedes most splicing factor recruitment and splicing. We are beginning to address these snRNP recruitment issues in metazoans, to compare them with yeast.
The recruitment of splicing factors and other mRNA-binding proteins to nascent mRNA likely involves RNA polymerase II and other elements of the transcriptional machinery, as well as chromatin. Three recent findings have heightened our interest in chromatin, nuclear mRNP, and the transcription machinery. First, a genetic screen implicates the transcription machinery in splicing efficiency. Second, we have purified active RNA polymerase II from chromatin; RNP proteins are copurified, indicating an association of these components with elongating polymerase. We are also trying to replicate these approaches in metazoans. Third, we have recently completed a different kind of genetic screen that implicates chromatin-associated mRNP and polyadenylation efficiency in tethering active genes to the nuclear periphery. (A grant from the National Institutes of Health provided support for these gene expression and yeast projects.)
Rhythms and Behavior
When we began our studies of Drosophila circadian rhythms more than 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. In 1990, we discovered that per mRNA as well as its encoded protein (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 many additional clock components defined by genetics in Drosophila over the past decade or so are largely conserved with mammals and perform similar functions in the mammalian clock. This indicates that the machinery as well as the principles of the Drosophila clock is widely conserved.
Our current circadian work has three goals: (1) to understand in biochemical detail how the Drosophila circadian clock functions (for example, how does the clock keep 24-hour time?), (2) to understand the neural circuit(s) relevant to circadian timekeeping within the fruit fly brain and the functions of individual circadian neurons, and (3) to understand the relationship of circadian timekeeping and circuits to the Drosophila sleep-wake cycle.
Although transcription factors and transcriptional regulation play key roles in circadian rhythmicity, post-transcriptional regulation has received recent, prominent attention. This is due to the potent effect of kinase mutants on the Drosophila clock, as well as some remarkable results from the cyanobacterial circadian system. Nonetheless, our results on the first goal described above continue to reinforce the importance of transcriptional regulation and the heterodimeric transcription factors Clock (CLK) and Cycle (CYC). This key complex drives the transcription of per and tim as well as the circadian transcription of other direct target genes. One of these is clockwork orange; CWO contributes to feedback repression and complements the role of PER-TIM in the feedback inhibition of CLK-CYC activity. Nonetheless, the temporal dynamics of this feedback and how it is regulated are still not understood. To gain further insight into the timing mechanisms, we continue to use a variety of biochemical and genetic strategies to alter transcriptional or post-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. The relationship between the two oscillators can switch as a function of environmental conditions, and the evening cells are the masters in constant light. Inter-oscillator communication may therefore serve in part as a seasonal timer, to adjust locomotor activity rhythms to day-length changes. Not surprisingly, recent results suggest that the different circadian cells have distinct relationships to light, with one set of cells tuned to dawn and another tuned to dusk. The role of light and its relationship to circadian cells also impact on fly arousal and sleep, the third goal described above. To extend these analyses of neuronal function, circuitry, and sleep-wake, we are developing new assays to visualize transcriptional oscillations as well as calcium levels in clock neurons and circuits in real time.
The increasing awareness of neuronal specificity with the circadian network also offers an opportunity for gene discovery. We have begun to profile mRNAs within circadian neuronal subtypes. Although core clock genes are highly enriched in all of these brain neurons, many mRNAs appear to be enriched in a neuron-specific manner, which may provide a link to the distinct functions of different circadian cells. The differentially expressed mRNAs should also provide invaluable tools for manipulating these different circadian neurons.
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 June 12, 2008
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