Transcriptional feedback and definition of the circadian pacemaker in Drosophila and animals.

The modern era of Drosophila circadian rhythms began with the landmark Benzer and Konopka paper and its definition of the period gene. The recombinant DNA revolution then led to the cloning and sequencing of this gene. This work did not result in a coherent view of circadian rhythm biochemistry, but experiments eventually gave rise to a transcription-centric view of circadian rhythm generation. Although these circadian transcription-translation feedback loops are still important, their contribution to core timekeeping is under challenge. Indeed, kinases and posttranslational regulation may be more important, based in part on recent in vitro work from cyanobacteria. In addition, kinase mutants or suspected kinase substrate mutants have unusually large period effects in Drosophila. This chapter discusses our recent experiments, which indicate that circadian transcription does indeed contribute to period determination in this system. We propose that cyanobacteria and animal clocks reflect two independent origins of circadian rhythms.

Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing.

Here we investigate the promoter control of alternative splicing by studying two transcriptional activators on templates under replicating conditions. SV40 large T-antigen (T-Ag) activates template replication only 2-fold but transcription 25-fold. T-Ag-mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10- to 30-fold increase in the inclusion of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T-Ag and VP16 promote conspicuous co-localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co-localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.

Coordination between transcription and pre-mRNA processing.

A large body of work has proved that transcription by RNA polymerase II and pre-mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy-terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing.

Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.