Splicing and transcription touch base: co-transcriptional spliceosome assembly and function.

Several macromolecular machines collaborate to produce eukaryotic messenger RNA. RNA polymerase II (Pol II) translocates along genes that are up to millions of base pairs in length and generates a flexible RNA copy of the DNA template. This nascent RNA harbours introns that are removed by the spliceosome, which is a megadalton ribonucleoprotein complex that positions the distant ends of the intron into its catalytic centre. Emerging evidence that the catalytic spliceosome is physically close to Pol II in vivo implies that transcription and splicing occur on similar timescales and that the transcription and splicing machineries may be spatially constrained. In this Review, we discuss aspects of spliceosome assembly, transcription elongation and other co-transcriptional events that allow the temporal coordination of co-transcriptional splicing.

Assembly and function of the amyloid-like translational repressor Rim4 is coupled with nutrient conditions.

Amyloid-like protein assemblies have been associated with toxic phenotypes because of their repetitive and stable structure. However, evidence that cells exploit these structures to control function and activity of some proteins in response to stimuli has questioned this paradigm. How amyloid-like assembly can confer emergent functions and how cells couple assembly with environmental conditions remains unclear. Here, we study Rim4, an RNA-binding protein that forms translation-repressing assemblies during yeast meiosis. We demonstrate that in its assembled and repressive state, Rim4 binds RNA more efficiently than in its monomeric and idle state, revealing a causal connection between assembly and function. The Rim4-binding site location within the transcript dictates whether the assemblies can repress translation, underscoring the importance of the architecture of this RNA-protein structure for function. Rim4 assembly depends exclusively on its intrinsically disordered region and is prevented by the Ras/protein kinase A signaling pathway, which promotes growth and suppresses meiotic entry in yeast. Our results suggest a mechanism whereby cells couple a functional protein assembly with a stimulus to enforce a cell fate decision.

A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.

openBIS ELN-LIMS: an open-source database for academic laboratories.

UNLABELLED: The open-source platform openBIS (open Biology Information System) offers an Electronic Laboratory Notebook and a Laboratory Information Management System (ELN-LIMS) solution suitable for the academic life science laboratories. openBIS ELN-LIMS allows researchers to efficiently document their work, to describe materials and methods and to collect raw and analyzed data. The system comes with a user-friendly web interface where data can be added, edited, browsed and searched. AVAILABILITY AND IMPLEMENTATION: The openBIS software, a user guide and a demo instance are available at https://openbis-eln-lims.ethz.ch. The demo instance contains some data from our laboratory as an example to demonstrate the possibilities of the ELN-LIMS (Ottoz et al., 2014). For rapid local testing, a VirtualBox image of the ELN-LIMS is also available.

Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae.

The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone ß-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with ß-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.

The role of disorder in RNA binding affinity and specificity.

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA-protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein-protein and RNA-protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.

Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells.

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.