Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogasterAdh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability.

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 min - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex.

Nuclear pore complexes (NPCs) are ∼100 MDa transport channels assembled from multiple copies of ∼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture.

ATPase activity of the DEAD-box protein Dhh1 controls processing body formation.

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4.

For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother-bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.

Spindle position is coordinated with cell-cycle progression through establishment of mitotic exit-activating and -inhibitory zones.

How spatial information is translated into a chemical signal is a fundamental problem in all organisms. The spindle position checkpoint is a prime example of this problem. This checkpoint senses spindle position and, in budding yeast, inhibits the mitotic exit network (MEN), a signaling pathway that promotes exit from mitosis. We find that spindle position is sensed by a system composed of MEN-inhibitory and -activating zones and a sensor that moves between them. The MEN inhibitory zone is located in the mother cell, the MEN-activating zone in the bud, and the spindle pole body (SPB), where the components of the MEN reside, functions as the sensor. Only when an SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud where the MEN activator Lte1 resides can exit from mitosis occur. In this manner, spatial information is sensed and translated into a chemical signal.

The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

Effects of aneuploidy on cellular physiology and cell division in haploid yeast.

Aneuploidy is a condition frequently found in tumor cells, but its effect on cellular physiology is not known. We have characterized one aspect of aneuploidy: the gain of extra chromosomes. We created a collection of haploid yeast strains that each bear an extra copy of one or more of almost all of the yeast chromosomes. Their characterization revealed that aneuploid strains share a number of phenotypes, including defects in cell cycle progression, increased glucose uptake, and increased sensitivity to conditions interfering with protein synthesis and protein folding. These phenotypes were observed only in strains carrying additional yeast genes, which indicates that they reflect the consequences of additional protein production as well as the resulting imbalances in cellular protein composition. We conclude that aneuploidy causes not only a proliferative disadvantage but also a set of phenotypes that is independent of the identity of the individual extra chromosomes.

Refactoring bacteriophage T7.

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11,515 base pairs (bp) of the 39,937 bp wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.