Intrinsically disordered signaling proteins: Essential hub players in the control of stress responses in Saccharomyces cerevisiae.

Cells have developed diverse mechanisms to monitor changes in their surroundings. This allows them to establish effective responses to cope with adverse environments. Some of these mechanisms have been well characterized in the budding yeast Saccharomyces cerevisiae, an excellent experimental model to explore and elucidate some of the strategies selected in eukaryotic organisms to adjust their growth and development in stressful conditions. The relevance of structural disorder in proteins and the impact on their functions has been uncovered for proteins participating in different processes. This is the case of some transcription factors (TFs) and other signaling hub proteins, where intrinsically disordered regions (IDRs) play a critical role in their function. In this work, we present a comprehensive bioinformatic analysis to evaluate the significance of structural disorder in those TFs (170) recognized in S. cerevisiae. Our findings show that 85.2% of these TFs contain at least one IDR, whereas ~30% exhibit a higher disorder level and thus were considered as intrinsically disordered proteins (IDPs). We also found that TFs contain a higher number of IDRs compared to the rest of the yeast proteins, and that intrinsically disordered TFs (IDTFs) have a higher number of protein-protein interactions than those with low structural disorder. The analysis of different stress response pathways showed a high content of structural disorder not only in TFs but also in other signaling proteins. The propensity of yeast proteome to undergo a liquid-liquid phase separation (LLPS) was also analyzed, showing that a significant proportion of IDTFs may undergo this phenomenon. Our analysis is a starting point for future research on the importance of structural disorder in yeast stress responses.

High-throughput replica-pinning approach to screen for yeast genes controlling low-frequency events.

Saccharomyces cerevisiae is a leading model system for genome-wide screens, but low-frequency events (e.g., point mutations, recombination events) are difficult to detect with existing approaches. Here, we describe a high-throughput screening technique to detect low-frequency events using high-throughput replica pinning of high-density arrays of yeast colonies. This approach can be used to screen genes that control any process involving low-frequency events for which genetically selectable reporters are available, e.g., spontaneous mutations, recombination, and transcription errors. For complete details on the use and execution of this protocol, please refer to (Novarina et al., 2020a, 2020b).

The canonical RdDM pathway mediates the control of seed germination timing under salinity.

Plants respond to adverse environmental cues by adjusting a wide variety of processes through highly regulated mechanisms to maintain plant homeostasis for survival. As a result of the sessile nature of plants, their response, adjustment and adaptation to the changing environment is intimately coordinated with their developmental programs through the crosstalk of regulatory networks. Germination is a critical process in the plant life cycle, and thus plants have evolved various strategies to control the timing of germination according to their local environment. The mechanisms involved in these adjustment responses are largely unknown, however. Here, we report that mutations in core elements of canonical RNA-directed DNA methylation (RdDM) affect the germination and post-germination growth of Arabidopsis seeds grown under salinity stress. Transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses support the involvement of this pathway in the control of germination timing and post-germination growth under salinity stress by preventing the transcriptional activation of genes implicated in these processes. Subsequent transcriptional effects on genes that function in relation to these developmental events support this conclusion.

Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D-cyclin-dependent kinase complexes.

The family of maize Kip-related proteins (KRPs) has been studied and a nomenclature based on the relationship to rice KRP genes is proposed. Expression studies of KRP genes indicate that all are expressed at 24 h of seed germination but expression is differential in the different tissues of maize plantlets. Recombinant KRP1;1 and KRP4;2 proteins, members of different KRP classes, were used to study association to and inhibitory activity on different maize cyclin D (CycD)-cyclin-dependent kinase (CDK) complexes. Kinase activity in CycD2;2-CDK, CycD4;2-CDK, and CycD5;3-CDK complexes was inhibited by both KRPs; however, only KRP1;1 inhibited activity in the CycD6;1-CDK complex, not KRP4;2. Whereas KRP1;1 associated with either CycD2;2 or CycD6;1, and to cyclin-dependent kinase A (CDKA) recombinant proteins, forming ternary complexes, KRP4;2 bound CDKA and CycD2;2 but did not bind CycD6;1, establishing a differential association capacity. All CycD-CDK complexes included here phosphorylated both the retinoblastoma-related (RBR) protein and the two KRPs; interestingly, while KRP4;2 phosphorylated by the CycD2;2-CDK complex increased its inhibitory capacity, when phosphorylated by the CycD6;1-CDK complex the inhibitory capacity was reduced or eliminated. Evidence suggests that the phosphorylated residues in KRP4;2 may be different for every kinase, and this would influence its performance as a cyclin-CDK inhibitor.