Papillary thyroid cancer (PTC) is one of the most prevalent endocrine malignancies and is associated with severe morbidity and high mortality. This study aimed to explore the role of long non-coding RNA (lncRNA) SLC8A1 antisense RNA 1 (SLC8A1-AS1) in the pathogenesis of PTC. In this study, we explored the function of SLC8A1-AS1 in PTC progression. We observed that the expression of SLC8A1-AS1 was downregulated in clinical PTC samples and PTC cell lines compared to that in normal controls. Cell counting kit (CCK)-8 assays demonstrated that the overexpression of SLC8A1-AS1 significantly reduced the proliferation of PTC cells. Consistently, apoptosis of PTC cells was enhanced by SLC8A1-AS1 overexpression. SLC8A1-AS1 overexpression attenuated the invasion and migration of PTC cells. Mechanistically, SLC8A1-AS1 maintained NUMB like endocytic adaptor protein (Numbl) mRNA stability by interacting with FUS RNA Binding Protein (FUS) in PTC cells. Depletion of Numbl reversed the inhibitory effect of SLC8A1-AS1 overexpression on PTC. Thus, we concluded that SLC8A1-AS1 suppresses PTC progression via the FUS/Numbl axis. Our findings provide novel insights into the mechanism underlying SLC8A1-AS1 attenuation of the malignant development of PTC, improving our understanding of the association between lncRNAs and PTC. SLC8A1-AS1 and FUS may be potential targets for PTC treatment.

RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. In one such pathway the tumor-suppressor protein adenomatous polyposis coli (APC) targets RNAs to cell protrusions, forming APC-containing ribonucleoprotein complexes (APC-RNPs). Here, we show that APC-RNPs associate with the RNA-binding protein Fus/TLS (fused in sarcoma/translocated in liposarcoma). Fus is not required for APC-RNP localization but is required for efficient translation of associated transcripts. Labeling of newly synthesized proteins revealed that Fus promotes translation preferentially within protrusions. Mutations in Fus cause amyotrophic lateral sclerosis (ALS) and the mutant protein forms inclusions that appear to correspond to stress granules. We show that overexpression or mutation of Fus results in formation of granules, which preferentially recruit APC-RNPs. Remarkably, these granules are not translationally silent. Instead, APC-RNP transcripts are translated within cytoplasmic Fus granules. These results unexpectedly show that translation can occur within stress-like granules. Importantly, they identify a new local function for cytoplasmic Fus with implications for ALS pathology.

The endothelium is crucially involved in the pathogenesis of atherosclerosis according to accumulating evidence. Moreover, recent studies have showed that lncRNAs could serve as biomarkers of cardiovascular diseases, in particular atherosclerosis. However, the underlying mechanism of endothelial dysfunction involving lncRNAs in atherosclerosis remains unknown. This study investigated the mechanism of lncRNA XXYLT1-AS2 in endothelial dysfunction in atherosclerosis.

FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kd (app) values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have been linked to severe neurodegenerative diseases; hence, it is of great interest to understand this process and how it is dysregulated in disease. Transportin-1 (TNPO1) and the closely related transportin-2 have been identified as major nuclear import receptors of FUS. They bind to the C-terminal nuclear localization signal of FUS and mediate the protein's nuclear import and at the same time also suppress aberrant phase transitions of FUS in the cytoplasm. Whether FUS can utilize other nuclear transport receptors for the purpose of import and chaperoning has not been examined so far. Here, we show that FUS directly binds to different import receptors in vitro. FUS formed stable complexes not only with TNPO1 but also with transportin-3, importin β, importin 7, or the importin β/7 heterodimer. Binding of these alternative import receptors required arginine residues within FUS-RG/RGG motifs and was weakened by arginine methylation. Interaction with these importins suppressed FUS phase separation and reduced its sequestration into stress granules. In a permeabilized cell system, we further showed that transportin-3 had the capacity to import FUS into the nucleus, albeit with lower efficiency than TNPO1. Our data suggest that aggregation-prone RNA-binding proteins such as FUS may utilize a network of importins for chaperoning and import, similar to histones and ribosomal proteins.

Unexplained recurrent pregnancy loss (URPL) is a significant reproductive health issue, affecting approximately 5% of pregnancies. Enhancer RNAs (eRNAs) have been reported to play important roles during embryo development and may be related to URPL. To investigate whether and how eRNAs are involved in URPL, we performed RNA sequencing in decidual tissue. Through comprehensive screening and validation, we identified a decidua-enriched eRNA long noncoding-CES1-1 (lnc-CES1-1) enriched in URPL patients and studied its function in decidua-associated cell lines (DACs). Higher expression of lnc-CES1-1 increased the level of inflammatory factors tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) and impaired the cell migration ability, which was attenuated by downregulating peroxisome proliferators-activated receptor γ (PPARγ). Upon activation by signal transduction and activation of transcription 4 (STAT4), lnc-CES1-1 interacted with the transcription factor fused in sarcoma (FUS) to upregulate the expression of PPARγ and affected cell migration. Taken together, these findings provide novel insights into the biological functions of decidua-associated lnc-CES1-1 and the molecular mechanisms underlying URPL. Our findings indicated that lnc-CES1-1 might be a potential candidate biomarker for URPL diagnosis and treatment.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community.  In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif of FUS as a key driver of condensate aging. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors has one of the worst prognoses, and the role of long noncoding RNAs (lncRNAs) in the biological and pathological processes of pancreatic cancer, including tumor cell proliferation, is a popular topic in tumor research. Our previous study revealed the correlation between high levels of the lncRNA-SOX2OT (SOX2OT) with poor survival outcomes. Cell Counting Kit-8, EdU, Flow cytometry and Colony formation assays as well as Xenograft growth of PDAC cells in mice were used for the detection of PDAC cells proliferation progression. Fluorescence in situ hybridization, RNA-binding protein pulldown and RNA immunoprecipitation assays were also used to identify the putative mechanisms of SOX2OT participating in the tumor progression. SOX2OT and its potential downstream targets were verified by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). SOX2OT was confirmed to promote the proliferation of PDAC cells. It was found to directly physically bind to FUS and we also demonstrated that FUS protein stability was affected by binding with SOX2OT and FUS could suppressed PDAC tumor by regulating cell cycle-associated factors CCND1 and p27. Our findings suggest that SOX2OT may act as a tumor promoter in PDAC through physically binding FUS and regulating its downstream cell cycle-associated factors CCND1 and p27. It may serve as an effective target for antitumor treatment for pancreatic cancer.

Genes encoding RNA-binding proteins, including FUS and TDP43, play a central role in different neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recently, a mutation located in the nuclear export signal (NES) of the FUS gene has been reported to cause an autosomal dominant form of familial Essential tremor.

TDP-43 and FUS are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and loss of function of either protein contributes to these neurodegenerative conditions. To elucidate the TDP-43- and FUS-regulated pathophysiological RNA metabolism cascades, we assessed the differential gene expression and alternative splicing profiles related to regulation by either TDP-43 or FUS in primary cortical neurons. These profiles overlapped by >25% with respect to gene expression and >9% with respect to alternative splicing. The shared downstream RNA targets of TDP-43 and FUS may form a common pathway in the neurodegenerative processes of ALS/FTLD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobe degeneration (FTLD) are two inter-related intractable diseases of motor neuron degeneration. Fused in sarcoma (FUS) is found in cytoplasmic accumulation of ALS and FTLD patients, which readily link the protein with the diseases. The RNA recognition motif (RRM) of FUS has the canonical α-β folds along with an unusual lysine-rich loop (KK-loop) between α1 and β2. This KK-loop is highly conserved among FET family proteins. Another contrasting feature of FUS RRM is the absence of critical binding residues, which are otherwise highly conserved in canonical RRMs. These residues in FUS RRM are Thr286, Glu336, Thr338, and Ser367, which are substitutions of lysine, phenylalanine, phenylalanine, and lysine, respectively, in other RRMs. Considering the importance of FUS in RNA regulation and metabolism, and its implication in ALS and FTLD, it is important to elucidate the underlying molecular mechanism of RNA recognition. In this study, we have performed molecular dynamics simulation with enhanced sampling to understand the conformational dynamics of noncanonical FUS RRM and its binding with RNA. We studied two sets of mutations: one with alanine mutation of KK-loop and another with KK-loop mutations along with critical binding residues mutated back to their canonical form. We find that concerted movement of KK-loop and loop between β2 and β3 facilitates the folding of the partner RNA, indicating an induced-fit mechanism of RNA binding. Flexibility of the RRM is highly restricted upon mutating the lysine residues of the KK-loop, resulting in weaker binding with the RNA. Our results also suggest that absence of the canonical residues in FUS RRM along with the KK-loop is equally important in regulating its binding dynamics. This study provides a significant structural insight into the binding of FUS RRM with its cognate RNA, which may further help in designing potential drugs targeting noncanonical RNA recognition.

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Pancreatic cancer is a highly aggressive and lethal digestive system malignancy. Our previous studies revealed the correlation of high levels of lncRNA SOX2OT expression with patients' poor survival outcomes, the promoting role of SOX2OT in proliferation and cycle progression of pancreatic cancer cells, and the in vivo binding of SOX2OT to RNA binding protein FUS, which destabilized the protein expression of FUS. However, the mechanism of SOX2OT binding and inhibiting FUS protein stability remains unclear. In this study, we performed RNA pull-down, cycloheximide-chase, and ubiquitination assays to determine the effect of SOX2OT on FUS ubiquitination, and explored the specific regulatory mechanism of SOX2OT-FUS axis in pancreatic cancer cell migration, invasion, in vivo tumor growth, and metastasis through RNA sequencing. We found that SOX2OT binds to FUS through its 5' and 3' regions, resulting in FUS ubiquitination and degradation. The SOX2OT-FUS regulatory axis promotes migration, invasion, tumor growth, and metastasis ability of pancreatic cancer cells. The in-depth elaboration of the SOX2OT-FUS regulatory axis in pancreatic cancer may clarify the mechanism of action of SOX2OT and provide new ideas for pancreatic cancer treatment.