Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

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Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure [] (reviewed in [, , ]). Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores [, , , ]. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs [, ]. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L [, ] and the core protein G3BP1 [, , , ] occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.

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Stress granules comprised of RNA (red) and protein assemblies (green) formed in part through RNA-RNA interactions.

“While proteins have long been recognized to form aberrant complexes that can trigger disease, RNA has generally not been thought to form promiscuous assemblies that might have functional roles in cells as well as cause problems in some contexts,” said Roy Parker, Distinguished Professor of Biochemistry at CU �鶹ӰԺ.

The study, published recently in Cell, highlights how energetically favorable RNA self-assembly is and identifies one way to actively prevent this assembly from growing out of control. Parker has long studied the properties of stress granules and has pioneered the model that RNA-RNA interactions are a significant contributor to stress granule assembly. 

This study, spearheaded by two brothers in the Parker research group at CU �鶹ӰԺ, presents two meaningful conclusions. First, stress granules and other ribonucleoprotein (RNP) complexes readily form favorable interactions with free RNAs. These interactions recruit new RNAs onto the surface of the RNP, thereby growing and stabilizing the complex. Second, a highly abundant enzyme within cells, known as eIF4A1, functions as an “RNA chaperone” to prevent the unregulated growth of RNPs within cells by binding to RNAs.

Devin (L) and Gabriel (R) Tauber, Parker Research Group, �鶹ӰԺ

Co-first authors and brothers Devin and Gabriel Tauber used their complementary expertise in Parker’s lab to understand RNA recruitment to RNPs both in a test tube and in living cells. Devin is a Ph.D. student, and Gabriel was an undergraduate at the time of the study. While unplanned, the brotherly collaboration resulted in an elegant characterization of RNA self-assembly and uncovered the role of the enzyme eIF4A1 in limiting this process in cells. 

“Gabe and I have always been interested in science, but we never thought we’d publish a research article together, let alone work in the same lab. Yet, we both fell in love with RNA research and became engaged in understanding the many ways in which RNA can function in the cell beyond simply serving as a middle-man between DNA and protein synthesis,” said Devin. “Since we are brothers, when one of us comes up with an off the wall idea, we are comfortable letting each other have it without the risk of endangering a professional relationship.”

Gabriel sought to understand RNA recruitment into RNPs by watching fluorescently-labeled RNA species assemble under the microscope. Gabriel observed robust recruitment of RNAs with every type of RNP tested. This result raised the question – how do cells limit the growth of RNPs such as stress granules?

The authors believed that the enzyme eIF4A1 was the most likely mechanism to prevent aberrant RNA assembly. eIF4A1 is one of the most abundant RNA binding proteins in the cell and uses energy in the form of ATP to disrupt RNA-RNA interactions. Using fluorescence microscopy to view individual cells, they saw that eIF4A1 is concentrated at the periphery of stress granules, providing further support for the idea that eIF4A1 disrupts RNA-RNA interactions at the surface of RNPs. Thus, Devin sought out to ask whether modulating the levels of eIF4A1 in the cell would affect stress granule assembly.

The Tauber brothers observed that depleting the cell of eIF4A1 can induce stress granule assembly under conditions where they typically do not form. Conversely, they found that increasing the amount of eIF4A1 in the cell is sufficient to prevent stress granule formation under conditions where they would normally develop. However, a mutant form of eIF4A1 which cannot bind to RNA was unable to repress stress granule formation. Together, these experiments solidified the role of eIF4A1 as an inhibitor of RNA recruitment to stress granules and helped to shape the model of RNP assembly as a highly favorable process which requires the cell to use energy to limit it.

“This work will trigger a new set of studies on understanding how cells control RNA-RNA interactions to keep RNAs in the proper balance between functional and specific interactions while limiting inappropriate interactions,” said Parker.

eIF4A’s “RNA chaperone” function could be considered analogous to heat shock proteins, which prevent protein aggregation by binding to unfolded proteins. Protein aggregates that may contain RNA are commonly found in neurodegenerative diseases such as Alzheimer’s disease and Amyotrophic lateral sclerosis. Identifying the respective roles of RNA and protein in the formation of these aggregates could provide critical insight into the cause of these diseases.

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Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response since many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown.  Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, as RLBs did not require protein kinase R (PKR), phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

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The proper regulation of mRNA processing, localization, translation, and degradation occurs on mRNPs. However, the global principles of mRNP organization are poorly understood. Although much information remains to be discovered, we utilize existing information to present a synthesis of mRNP understanding with the following key points. First, mRNPs form a compacted structure due to the inherent folding of RNA. Second, the ribosome is the principal mechanism by which mRNA regions are partially decompacted. Third, mRNPs are 50%-80% protein by weight, suggesting the majority of mRNA sequences are not directly interacting with RNA binding proteins. Finally, the ratio of mRNA-binding proteins to mRNAs is higher in the nucleus to allow effective RNA processing and limit the potential for nuclear RNA based aggregation. This synthesis of mRNP understanding provides a model for mRNP biogenesis, structure, and regulation with multiple implications.

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RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don’t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

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Toxic protein assemblies, or "amyloids," long considered to be key drivers in many neuromuscular diseases, also play a beneficial role in the development of healthy muscle tissue, �鶹ӰԺ researchers have found.

"Ours is the first study to show that amyloid-like structures not only exist in healthy skeletal muscle during regeneration, but are likely important for its formation," said co-first author Thomas Vogler, an M.D./PhD candidate in the Department of Molecular, Cellular, and Developmental Biology (MCDB).

The surprising finding, published today in the journal Nature, sheds new light on the potential origins of a host of incurable disorders, ranging from amyotrophic lateral sclerosis (ALS) to inclusion body myopathy (which causes debilitating muscle degeneration) to certain forms of muscular dystrophy.

The researchers believe it could ultimately open new avenues for treating musculoskeletal diseases and also lend new understanding to related neurological disorders like Parkinson's and Alzheimer's disease, in which different amyloids play a role.

"Many of these degenerative diseases share a similar scenario in which they have these protein aggregates that accumulate in the cell and gum up the system," said co-first author Joshua Wheeler, also an M.D./PhD candidate in the Department of Biochemistry. "As these aggregates are beneficial for normal regeneration, our data suggest that the cell is just damaged and trying to repair itself."

For the study, Vogler and MCDB professor Brad Olwin, who study muscle generation, teamed up with Wheeler and Roy Parker, who study RNA, to investigate a protein called TDP-43.

TDP-43 has long been suspected to be a culprit in disease, having been found in the skeletal muscle of people with inclusion body myopathy and the neurons of people with ALS. But when the researchers closely examined muscle tissue growing in culture in the lab, they discovered clumps of TDP-43 were present not only in diseased tissue but also in healthy tissue.

"That was astounding," said Olwin. "These amyloid-like aggregates, which we thought were toxic, seemed to be a normal part of muscle formation, appearing at a certain time and then disappearing again once the muscle was formed."

Subsequent studies in muscle tissue growing in culture showed that when the gene that codes for TDP-43 was knocked out, muscles didn't grow. When the researchers looked at human tissue biopsied from healthy people whose muscles were regenerating, they found aggregates, or "myo-granules," of TDP-43. Further RNA-protein mapping analysis showed that the clusters - like shipping trucks traveling throughout the cell - appear to carry instructions for how to build contractile muscle fibers.

Wheeler and Vogler, both competitive runners and long-time friends, came up with the initial idea for the study while on a trail run. Wheeler says the data suggest that when healthy athletes push their muscles hard via things like marathons and ultramarathons, they are probably also forming amyloid-like clusters within their cells.

The key question remains: Why do most people quickly clear these proteins while others do not, with. the granules - like sugar cubes that won't dissolve - clustering together and causing disease?

"If they normally form and go away, something is making them dissolve," said Olwin. "Figuring out the mechanisms involved could potentially open a new avenue for treatments."

The team is also interested in exploring whether a similar process may occur in the brain after injury, kick-starting disease. And subsequent studies will go even further to identify what the protein clusters do.

"This is a great example of how collaboration across disciplines can lead to really important work," said Parker.

As participants in CU's Medical Science Training Program, which enables students to concurrently pursue a medical degree at the Anschutz Medical Campus and a PhD at CU �鶹ӰԺ, Wheeler and Vogler hope that someday the work they do in the lab will help the patients they see in the clinic.

"The holy grail of all this is to be able to treat devastating and incurable diseases like ALS and to develop therapeutic strategies to improve skeletal muscle and fitness," said Wheeler. "We are just opening the door on this."

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With approval Sept. 14 by the Board of Regents, the University of Colorado has introduced seven newly designated distinguished professors, six of whom are affiliated with the CU �鶹ӰԺ campus.

They are Natalie Ahn, James Anaya, Elizabeth Fenn, John Lynch, Warren Motte and Roy Parker. From the CU Anschutz Medical Campus, Larry Green also was designated a distinguished professor. 

Distinguished professors are faculty members who demonstrate exemplary performance in research or creative work, a record of excellence in classroom teaching and supervision of individual learning, and outstanding service to the profession, the university and its affiliates.

The CU campuses nominate faculty for the award, the highest honor bestowed upon faculty across the system's four campuses. President Benson then reviews the nominations and, with the recommendation of the Distinguished Professors Committee, forwards the candadates' names to the Board of Regents. 

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