IMAGE: High-resolution confocal microscopy images of mCherry tagged NELF-A proteins (red) in human HeLa cells. The NELF-A wild type protein produces stress condensation on heat shock (left) but proteins with IDR deficiency fail … view more
Credit: © MPI of Immunbiology and Epigenetic, P. Rawat
All life on earth grew through layers and networks to ensure their survival at catastrophic events. Cells even have the emergency plan: the heat shock response. Motivated by multiple stress stimuli such as heat, toxins, or radiation, this cell safety program seeks to prevent permanent damage to the organism. The response is similar to an overall “locking” strategy adopted due to corona virus pandemic. At the time of lockdown, only necessary actions are allowed and resources have been shifted towards measures ensuring that the impact of pandemic disease is minimized.
Under normal conditions, RNA polymerase II degrades the DNA. At the right places, the DNA is transcribed into mRNA, which is then converted into proteins. In emergencies, however, this transcriptional activity must be stopped, in the main, in order to shut down or reduce unnecessary proteins at times of stress. “This move unleashes essential capabilities to ramp up the production of RNA and proteins called molecular chaperones, which help address the threat and effects of stress. The question remains, how to are you a completely locked cell? “said Ritwick Sawarkar, group director at MPI Immunobiology and Epigenetics and Cambridge University.
NELF density by weight – ensures reduced gene transcription
Earlier studies by the Sawarkar laboratory gave the first glimpse of what happens in the cells, when they move from normal to emergency. Stress causes accumulation of the negative elongation factor (NELF) in the nucleus and stops the transcription of a large number of genes. But what exactly is the execution of the NELF transcriptional regulator is called Stress-Induced Transcriptional Attenuation (SITA).
“At the beginning of this project, we tried to visualize NELF proteins with living cell images to better understand its function and regulation. Surprisingly, we found that NELF creates a punctate or droplets on weight but the same protein is still released under any pressure conditions. These droplets are called NELF condensates, “said Prashant Rawat, the study ‘s first author. Together with the MPI’s Patrick Cramer Lab for Biophysical Chemistry that could rebalance the same NELF droplets in vitro with reconstituted pure proteins, the teams suggest that the stress-induced biomolecular density enables better recruitment of NELF to gene-stimulated regions. Here, the NELF droplets appear to inhibit the activity of the polymerase and direct the depletion of gene expression.
Tentacle NELF resolution
NELF subunits contain regions called disorder (IDRs). IDRs are components of proteins without a fixed structure and act as adhesives. Scientists Max Planck was able to show that interactions between the NELF adhesives are critical for density. “A lot of individual NELF molecules come together and their grips are firmly bonded together to form the droplet just like holding each other’s hands. But the biggest thing that put them off. Our concern is that IDRs are always in NELF as part of their structure but just under pressure, “says Prashant Rawat.
Using molecular and biochemical methods across genome and proteome, the team identified specific Post-translational Modifications (PTMs) required for NELF density. PTMs are modifications of proteins after synthesis and are often used by cells to respond to environmental stimuli. The results show that two different modifications make NELF condensates possible. “We found that stress-induced changes in NELF phosphorylation and further SUMOylation regulate NELF density,” says Ritwick Sawarkar.
Relevant NELF concordance for cell fitness
Cells that do not produce NELF droplets due to lack of IDR or lack of SUMOylation also fail the genes and their transcription of stress. “If cells do not lock in with NELF density and transcription decline there is a risk that they will be unhealthy. Our data show significantly higher mortality rates of cells that do not have proper NELF density during stress , “said Prashant Rawat.
For Ritwick Sawarkar, these results also clarify the collaborative aspects of life at the Max Planck Institutes. “This research was made possible only due to close collaboration. Andrea Pichler’s lab at MPI-IE was crucial in understanding the role of the SUMO machine, and other collaborations with testing Patrick Cramer’s lab at MPI-BPC Göttingen rebalances the same NELF droplets in vitro with purified protein reabsorption, “said Ritwick Sawarkar, lead author of the study.
Hypertension-induced transcriptional depression is already thought to be associated with brain disorders such as Huntington’s. “We have already created mouse models at the institute to extend our results in vivo and to relevant disease models,” says Prashant Rawat. It seems possible to study the role of NELF condensates in various diseases as an interesting method for future laboratory research.
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