RESUMO
EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.
Assuntos
DNA , Estruturas R-Loop , Proteína EWS de Ligação a RNA , Proteína EWS de Ligação a RNA/metabolismo , Proteína EWS de Ligação a RNA/química , Proteína EWS de Ligação a RNA/genética , Humanos , DNA/metabolismo , DNA/química , Ligação Proteica , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/genética , Dedos de Zinco , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ensaio de Desvio de Mobilidade EletroforéticaRESUMO
EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.
RESUMO
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.
Assuntos
DNA , Estruturas R-Loop , Proteína FUS de Ligação a RNA , RNA , DNA/metabolismo , Estruturas R-Loop/genética , RNA/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Ligação Proteica , Humanos , RNA Polimerases Dirigidas por DNA/metabolismo , Células HEK293RESUMO
Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.
RESUMO
Purified recombinant FUsed in Sarcoma (FUS) assembles into an oligomeric state in an RNA-dependent manner to form large condensates. FUS condensates bind and concentrate the C-terminal domain of RNA polymerase II (RNA Pol II). We asked whether a granule in cells contained FUS and RNA Pol II as suggested by the binding of FUS condensates to the polymerase. We developed cross-linking protocols to recover protein particles containing FUS from cells and separated them by size exclusion chromatography. We found a significant fraction of RNA Pol II in large granules containing FUS with diameters of >50 nm or twice that of the RNA Pol II holoenzyme. Inhibition of transcription prevented the polymerase from associating with the granules. Altogether, we found physical evidence of granules containing FUS and RNA Pol II in cells that possess properties comparable to those of in vitro FUS condensates.
Assuntos
RNA Polimerase II/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Reagentes de Ligações Cruzadas , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Tamanho da Partícula , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/química , RNA Polimerase II/genética , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , RNA/metabolismo , Animais , Proteína do X Frágil da Deficiência Intelectual/química , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Quadruplex G , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Camundongos , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , RNA/química , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade EstáticaRESUMO
Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.
Assuntos
Fatores Quimiotáticos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Actinas/metabolismo , Quimiotaxia , Dictyostelium/citologia , Dictyostelium/efeitos dos fármacos , Modelos Biológicos , Miosinas/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Myofibrillar proteins must be removed from the myofibril before they can be turned over metabolically in functioning muscle cells. It is uncertain how this removal is accomplished without disruption of the contractile function of the myofibril. It has been proposed that the calpains could remove the outer layer of filaments from myofibrils as a first step in myofibrillar protein turnover. Several studies have found that myofilaments can be removed from myofibrils by trituration in the presence of ATP. These easily releasable myofilaments (ERMs) were proposed to be intermediates in myofibrillar protein turnover. It was unclear, however, whether the ERMs were an identifiable entity in muscle or whether additional trituration would remove more myofilaments until the myofibril was gone and whether calpains could release ERMs from intact myofibrils. The present study shows that few ERMs could be obtained from the residue after the first removal of ERMs, and the yield of ERMs from well-washed myofibrils was reduced, probably because some ERMs had been removed by the washing process. Mild calpain treatment of myofibrils released filaments that had a polypeptide composition and were ultrastructurally similar to ERMs. The yield of calpain-released ERMs was two- to threefold greater than the normal yield. Hence, ERMs are an identifiable entity in myofibrils, and calpain releases filaments that are similar to ERMs. The role of ERMs in myofibrillar protein turnover is unclear, because only filaments on the surface of the myofibril would turn over, and changes in myofibrillar protein isoforms during development could not occur via the ERM mechanism.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Calpaína/metabolismo , Bovinos , Músculo Esquelético/ultraestrutura , Miofibrilas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of mu-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the approximately 26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout mu-calpain and calpastatin were only partly purified; identity of trout mu-calpain was confirmed by labeling with antibodies to bovine skeletal muscle mu-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle mu- and m-calpain. Trout mu-calpain requires 4.4+/-2.8 microM and trout m-calpain requires 585+/-51 microM Ca(2+) for half-maximal activity, similar to the Ca(2+) requirements of mu- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.
Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/isolamento & purificação , Calpaína/metabolismo , Oncorhynchus mykiss , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Calpaína/genética , Bovinos , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Músculo Esquelético/química , Homologia de Sequência de AminoácidosRESUMO
Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).
Assuntos
Calpaína/química , Quimotripsina/química , Tripsina/química , Calpaína/metabolismo , Quimotripsina/metabolismo , Peptídeos/metabolismo , Fatores de Tempo , Tripsina/metabolismoRESUMO
The calpain system is involved in a number of human pathologies ranging from the muscular dystrophies to Alzheimer's disease. It is important, therefore, to be able to obtain and to characterize both mu-calpain and m-calpain from human tissue. Although human mu-calpain can be conveniently obtained from either erythrocytes or platelets, no readily available source of human m-calpain has been described. Human placenta extracts contain both mu-calpain and m-calpain in nearly equal proportions and in significant quantities (3-4 mg mu-calpain and 4-5 mg m-calpain/1000 g placenta tissue). Placenta also contains calpastatin that elutes off ion-exchange columns over a wide range of KCl concentrations completely masking the mu-calpain activity eluting off these columns and even partly overlapping m-calpain elution. Placenta mu-calpain requires 50-70 microM Ca2+ and placenta m-calpain requires 450-460 microM Ca2+ for half-maximal proteolytic activity. Western analysis of washed placenta tissue shows that placenta contains both mu- and m-calpain, although some of the mu-calpain in whole placenta extracts likely originates from the erythrocytes that are abundant in the highly vascularized placenta. Placenta calpastatin could not be purified with conventional methods. The most prominent form of calpastatin in Western analyses of placenta obtained as soon as possible after birth was approximately 48-51 kDa; partly purified preparations of placenta calpastatin also contained 48-51 and 70 kDa polypeptides. Human placenta extracts likely contain two different calpastatin isoforms, a 48-51 kDa "placenta calpastatin" and a 70 kDa erythrocyte calpastatin.
Assuntos
Calpaína/fisiologia , Placenta/fisiologia , Adulto , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/química , Calpaína/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , Feminino , Humanos , Isomerismo , Placenta/química , Placenta/metabolismo , Gravidez , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
It has been difficult to purify calpastatin without using a step involving heating to 90-100 degrees C. Preparations of calpastatin obtained after heating often contain several polypeptides that have been ascribed to proteolytic degradation. Because calpastatin is highly susceptible to proteolytic degradation and several different calpastatin isoforms can be produced by using different start sites of transcription/translation and/or alternative splicing from the single calpastatin gene, it is not clear whether the different polypeptides observed in purified calpastatin preparations are proteolytic fragments or calpastatin isoforms. It would be useful, therefore, to have a method for purifying calpastatin that does not involve heating. At low ionic strength, calpastatin from skeletal muscle extracts binds quantitatively to an immunoaffinity column made by coupling a monoclonal antibody (MAb) to the C-terminal end of calpastatin (epitope between amino acids 707 and 786) to agarose; the bound calpastatin can be eluted at pH 2.5. The C-terminal end of the calpastatin polypeptide was used because the known isoforms of calpastatin all contain domain IV. The eluted calpastatin, which retains all its calpain inhibitory activity, consists largely of a 125 kDa polypeptide (70%), and several smaller polypeptides that are labeled with a MAb to calpastatin. Expressed calpastatin constructs representing the full-length XL-IV calpastatin and domains L-IV, II-IV, III-IV, and IV also bind to the immunoaffinity column and can be purified. The immunoaffinity column is especially useful for purifying calpastatin from small tissue samples in a single step.