RESUMO
Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.
Assuntos
Proteínas de Transporte/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Autofagia/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Metionina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.
Assuntos
Proteínas de Transporte/metabolismo , Metionina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transição de Fase , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In this review, we describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. We propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity/intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-ß interactions that are both structurally specific and labile to disassembly. These weakly tethered assemblies, we propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.
Assuntos
Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Animais , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Expressão Gênica , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Hidrogéis , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
Assuntos
Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Arginina/química , Simulação por Computador , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Transição de Fase , Proteínas Priônicas/química , Proteínas Priônicas/genética , Príons/genética , Príons/fisiologia , Domínios Proteicos , Proteína FUS de Ligação a RNA/fisiologia , Proteínas de Ligação a RNA/isolamento & purificação , Células Sf9 , Tirosina/químicaRESUMO
Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.
Assuntos
Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear , Proteína FUS de Ligação a RNA/química , beta Carioferinas/química , Sítios de Ligação , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Carioferinas/metabolismo , Luz , Extração Líquido-Líquido , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Mutação , Nefelometria e Turbidimetria , Ligação Proteica , Domínios Proteicos , RNA/química , Espalhamento de Radiação , TemperaturaRESUMO
Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.
Assuntos
Proteína FUS de Ligação a RNA/química , Sequência de Aminoácidos , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Domínios Proteicos , Proteína FUS de Ligação a RNA/metabolismoRESUMO
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Grânulos Citoplasmáticos/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutagênese , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Prolina/análise , Prolina/metabolismo , Domínios Proteicos , Ribonucleases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Estresse FisiológicoRESUMO
This perspective begins with a speculative consideration of the properties of the earliest proteins to appear during evolution. What did these primitive proteins look like, and how were they of benefit to early forms of life? I proceed to hypothesize that primitive proteins have been preserved through evolution and now serve diverse functions important to the dynamics of cell morphology and biological regulation. The primitive nature of these modern proteins is easy to spot. They are composed of a limited subset of the 20 amino acids used by traditionally evolved proteins and thus are of low sequence complexity. This chemical simplicity limits protein domains of low sequence complexity to forming only a crude and labile type of protein structure currently hidden from the computational powers of machine learning. I conclude by hypothesizing that this structural weakness represents the underlying virtue of proteins that, at least for the moment, constitute the dark matter of the proteome.
Assuntos
Aminoácidos , Proteoma , Proteoma/química , Proteoma/metabolismo , Domínios Proteicos , Aminoácidos/metabolismoRESUMO
Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72 , Expansão das Repetições de DNA , Dipeptídeos/química , Dipeptídeos/genética , Demência Frontotemporal/genética , Células HeLa , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos/química , Peptídeos/genética , Domínios Proteicos , Proteínas/genéticaRESUMO
tRNA function is based on unique structures that enable mRNA decoding using anticodon trinucleotides. These structures interact with specific aminoacyl-tRNA synthetases and ribosomes using 3D shape and sequence signatures. Beyond translation, tRNAs serve as versatile signaling molecules interacting with other RNAs and proteins. Through evolutionary processes, tRNA fragmentation emerges as not merely random degradation but an act of recreation, generating specific shorter molecules called tRNA-derived small RNAs (tsRNAs). These tsRNAs exploit their linear sequences and newly arranged 3D structures for unexpected biological functions, epitomizing the tRNA "renovatio" (from Latin, meaning renewal, renovation, and rebirth). Emerging methods to uncover full tRNA/tsRNA sequences and modifications, combined with techniques to study RNA structures and to integrate AI-powered predictions, will enable comprehensive investigations of tRNA fragmentation products and new interaction potentials in relation to their biological functions. We anticipate that these directions will herald a new era for understanding biological complexity and advancing pharmaceutical engineering.
Assuntos
Aminoacil-tRNA Sintetases , RNA de Transferência , RNA de Transferência/metabolismo , Anticódon , Aminoacil-tRNA Sintetases/metabolismo , Ribossomos/metabolismo , RNA Mensageiro/genéticaRESUMO
Members of the FET protein family, consisting of FUS, EWSR1, and TAF15, bind to RNA and contribute to the control of transcription, RNA processing, and the cytoplasmic fates of messenger RNAs in metazoa. FET proteins can also bind DNA, which may be important in transcription and DNA damage responses. FET proteins are of medical interest because chromosomal rearrangements of their genes promote various sarcomas and because point mutations in FUS or TAF15 can cause neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. Recent results suggest that both the normal and pathological effects of FET proteins are modulated by low-complexity or prion-like domains, which can form higher-order assemblies with novel interaction properties. Herein, we review FET proteins with an emphasis on how the biochemical properties of FET proteins may relate to their biological functions and to pathogenesis.
Assuntos
Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Reparo do DNA , Humanos , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Processamento Pós-Transcricional do RNA , Proteína FUS de Ligação a RNA/química , Proteínas de Ligação a RNA/química , Fatores Associados à Proteína de Ligação a TATA/química , Transcrição GênicaRESUMO
Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.
Assuntos
Sarcoma de Ewing , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Mamíferos/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Ativação Transcricional/genéticaRESUMO
Amyloids are implicated in neurodegenerative and systemic diseases, yet they serve important functional roles in numerous organisms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that control central events of RNA biogenesis in normal and diseased cellular conditions. Many of these proteins contain prion-like sequences of low complexity, which not only assemble into functional fibrils in response to cellular cues but can also lead to disease when missense mutations arise in their sequences. Recent advances in cryo-electron microscopy (cryo-EM) have provided unprecedented high-resolution structural insights into diverse amyloid assemblies formed by hnRNPs and structurally related RBPs, including TAR DNA-binding protein 43 (TDP-43), Fused in Sarcoma (FUS), Orb2, hnRNPA1, hnRNPA2, and hnRNPDL-2. This review provides a comprehensive overview of these structures and explores their functional and pathological implications.
Assuntos
Amiloide , Proteínas de Ligação a RNA , Microscopia Crioeletrônica , Proteínas de Ligação a RNA/metabolismo , Amiloide/química , Amiloide/metabolismoRESUMO
Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing.
Assuntos
Arginina/metabolismo , Núcleo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Lisina/metabolismo , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Arginina/genética , Núcleo Celular/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Lisina/genética , Mutação , Fosforilação , Domínios Proteicos , RNA Mensageiro/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.
Assuntos
Proteínas de Drosophila/genética , Longevidade/genética , Fator 6 Associado a Receptor de TNF/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Enzimas Desubiquitinantes , Drosophila/genética , Longevidade/fisiologia , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ubiquitina/genéticaRESUMO
N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.
Assuntos
Adenosina/análogos & derivados , Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Precursores de RNA/biossíntese , RNA Mensageiro/biossíntese , Transcrição Gênica , Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Éxons , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Relação Estrutura-AtividadeRESUMO
"Complex multicellularity," conventionally defined as large organisms with many specialized cell types, has evolved five times independently in eukaryotes, but never within prokaryotes. A number of hypotheses have been proposed to explain this phenomenon, most of which posit that eukaryotes evolved key traits (e.g., dynamic cytoskeletons, alternative mechanisms of gene regulation, or subcellular compartments) which were a necessary prerequisite for the evolution of complex multicellularity. Here, we propose an alternative, nonadaptive hypothesis for this broad macroevolutionary pattern. By binning cells into groups with finite genetic bottlenecks between generations, the evolution of multicellularity greatly reduces the effective population size (Ne) of cellular populations, increasing the role of genetic drift in evolutionary change. While both prokaryotes and eukaryotes experience this phenomenon, they have opposite responses to drift: eukaryotes tend to undergo genomic expansion, providing additional raw genetic material for subsequent multicellular innovation, while prokaryotes generally face genomic erosion. Taken together, we hypothesize that these idiosyncratic lineage-specific evolutionary dynamics play a fundamental role in the long-term divergent evolution of complex multicellularity across the tree of life.
Assuntos
Evolução Biológica , Deriva Genética , Eucariotos/genética , Genoma , Regulação da Expressão GênicaRESUMO
Hox genes are important regulators in animal development. They often show a mosaic of conserved (e.g., longitudinal axis patterning) and lineage-specific novel functions (e.g., development of skeletal, sensory, or locomotory systems). Despite extensive research over the past decades, it remains controversial at which node in the animal tree of life the Hox cluster evolved. Its presence already in the last common metazoan ancestor has been proposed, although the genomes of both putative earliest extant metazoan offshoots, the ctenophores and the poriferans, are devoid of Hox sequences. The lack of Hox genes in the supposedly "simple"-built poriferans and their low number in cnidarians and the basally branching bilaterians, the xenacoelomorphs, seems to support the classical notion that the number of Hox genes is correlated with the degree of animal complexity. However, the 4-fold increase of the Hox cluster in xiphosurans, a basally branching chelicerate clade, as well as the situation in some teleost fishes that show a multitude of Hox genes compared to, e.g., human, demonstrates, that there is no per se direct correlation between organismal complexity and Hox number. Traditional approaches have tried to base homology on the morphological level on shared expression profiles of individual genes, but recent data have shown that, in particular with respect to Hox and other regulatory genes, complex gene-gene interactions rather than expression signatures of individual genes alone are responsible for shaping morphological traits during ontogeny. Accordingly, for sound homology assessments and reconstructions of character evolution on organ system level, additional independent datasets (e.g., morphological, developmental) need to be included in any such analyses. If supported by solid data, proposed structural homology should be regarded as valid and not be rejected solely on the grounds of non-parsimonious distribution of the character over a given phylogenetic topology.
Assuntos
Cnidários , Proteínas de Homeodomínio , Animais , Humanos , Filogenia , Proteínas de Homeodomínio/genética , Evolução Molecular , Cnidários/genética , Genes Homeobox/genética , Família Multigênica/genéticaRESUMO
Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly-with hardware requirements compatible with a laptop-models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.
Assuntos
Processamento Alternativo/genética , Proteômica , Splicing de RNA/genética , Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Anotação de Sequência Molecular , RNA Mensageiro/genética , TranscriptomaRESUMO
Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.