scholarly journals Droplet and fibril formation of the functional amyloid Orb2

2021 ◽  
Author(s):  
Kidist Ashami ◽  
Alexander S. Falk ◽  
Connor Hurd ◽  
Samridhi Garg ◽  
Silvia A. Cervantes ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Droplet Formation ◽  
Fibril Formation ◽  
Long Term Memory ◽  
Functional Amyloid

AbstractThe functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low complexity sequences similar to many RNA binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. We show that soluble Orb2 can indeed phase separate into protein droplets. However, these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. For Orb2 fragments that lack the C-terminal RNA binding domain (RBD), droplet formation is a prerequisite for fibril formation of an otherwise stable monomeric Orb2 solution. Solid-state NMR shows that these fibrils have additional well ordered static domains beside the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into cross-β fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.

scholarly journals SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

2020 ◽  
Author(s):  
Theodora Myrto Perdikari ◽  
Anastasia C. Murthy ◽  
Veronica H. Ryan ◽  
Scott Watters ◽  
Mandar T. Naik ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Host Protein ◽  
Host Cells ◽  
Liquid Liquid Phase Separation

AbstractTightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

scholarly journals FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair

2019 ◽  
Author(s):  
Brunno R. Levone ◽  
Silvia C. Lenzken ◽  
Marco Antonaci ◽  
Andreas Maiser ◽  
Alexander Rapp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Early Event ◽  
Structured Illumination Microscopy ◽  
Liquid Liquid Phase Separation

AbstractRNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, 53BP1, and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using super-resolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nano-foci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSBs repair complexes.

scholarly journals Low-complexity domain of U1-70K modulates phase separation and aggregation through distinctive basic-acidic motifs

2019 ◽  
Vol 5 (11) ◽  
pp. eaax5349 ◽  
Author(s):  
Song Xue ◽  
Rui Gong ◽  
Fanqi He ◽  
Yanqin Li ◽  
Yunjia Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Protein Domain ◽  
Shed Light ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) facilitates the formation of functional membraneless organelles and recent reports have linked this phenomenon to protein aggregation in neurodegenerative diseases. Understanding the mechanism of LLPS and its regulation thus promises to shed light on the pathogenesis of these conditions. The RNA-binding protein U1-70K, which aggregates in brains of Alzheimer’s disease patients, is considered a potential target for Alzheimer’s therapy. Here, we report that two fragments in the low-complexity (LC) domain of U1-70K can undergo LLPS. We have demonstrated that the repetitive basic-acidic motifs in these fragments induce nucleotide-independent phase separation and initiate aggregation in vitro. We also have confirmed that LLPS and aggregation occur in vivo and that the content of ampholytic motifs in a protein domain determines the transition between droplets and aggregation, providing insights into the mechanism underlying the formation of diverse assembly states.

scholarly journals FUS-dependent liquid–liquid phase separation is important for DNA repair initiation

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Brunno R. Levone ◽  
Silvia C. Lenzken ◽  
Marco Antonaci ◽  
Andreas Maiser ◽  
Alexander Rapp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Structured Illumination Microscopy ◽  
Liquid Liquid Phase Separation

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid–liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

scholarly journals PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

2018 ◽  
Author(s):  
Yongjia Duan ◽  
Aiying Du ◽  
Jinge Gu ◽  
Gang Duan ◽  
Chen Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Posttranslational Modifications ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Hnrnp A1 ◽  
Rnp Granules

SUMMARYMutations in RNA-binding proteins localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregations, which are pathological hallmarks in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of PARylation, an important PTM involved in DNA damage repair and cell death, in RNP-related neurodegeneration. We reveal that PARylation levels are a major regulator of the dynamic assembly-disassembly of RNP granules, and the disease-related RNPs such as hnRNP A1 and TDP-43 can both be PARylated and bind to PARylated proteins. We further identify the PARylation site of hnRNP A1 at K298, which controls the cytoplasmic translocation of hnRNP A1 in response to stress, as well as the PAR-binding motif (PBM) of hnRNP A1, which is required for the delivery and association of hnRNP A1 to stress granules. Moreover, we show that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, we establish that both genetic and pharmacological inhibition of PARP mitigates hnRNP A1 and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings indicate a novel and crucial role of PARylation in regulating the assembly and the dynamics of RNP granules, and dysregulation of PARylation may contribute to ALS disease pathogenesis.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

scholarly journals SSMART: Sequence-structure motif identification for RNA-binding proteins

2018 ◽  
Author(s):  
Alina Munteanu ◽  
Neelanjan Mukherjee ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Motif ◽  
Primary Sequence ◽  
Sequence Structure ◽  
Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

1996 ◽  
Vol 16 (7) ◽  
pp. 3668-3678 ◽  
Author(s):  
M F Henry ◽  
P A Silver
Keyword(s):  
Normal Cell ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Temperature Sensitive ◽  
Arginine Residues ◽  
Cognate Gene ◽  
Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.

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