RESUMO
In plants, miRNA production is orchestrated by a suite of proteins that control transcription of the pri-miRNA gene, post-transcriptional processing and nuclear export of the mature miRNA. Post-transcriptional processing of miRNAs is controlled by a pair of physically interacting proteins, hyponastic leaves 1 (HYL1) and Dicer-like 1 (DCL1). However, the evolutionary history and structural basis of the HYL1-DCL1 interaction is unknown. Here we use ancestral sequence reconstruction and functional characterization of ancestral HYL1 in vitro and in Arabidopsis thaliana to better understand the origin and evolution of the HYL1-DCL1 interaction and its impact on miRNA production and plant development. We found the ancestral plant HYL1 evolved high affinity for both double-stranded RNA (dsRNA) and its DCL1 partner before the divergence of mosses from seed plants (â¼500 Ma), and these high-affinity interactions remained largely conserved throughout plant evolutionary history. Structural modeling and molecular binding experiments suggest that the second of two dsRNA-binding motifs (DSRMs) in HYL1 may interact tightly with the first of two C-terminal DCL1 DSRMs to mediate the HYL1-DCL1 physical interaction necessary for efficient miRNA production. Transgenic expression of the nearly 200 Ma-old ancestral flowering-plant HYL1 in A. thaliana was sufficient to rescue many key aspects of plant development disrupted by HYL1- knockout and restored near-native miRNA production, suggesting that the functional partnership of HYL1-DCL1 originated very early in and was strongly conserved throughout the evolutionary history of terrestrial plants. Overall, our results are consistent with a model in which miRNA-based gene regulation evolved as part of a conserved plant "developmental toolkit."
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genéticaRESUMO
MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are â¼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.
Assuntos
MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Ribonuclease III/metabolismo , Sítio de Iniciação de Transcrição , Animais , Linhagem Celular , Guanina/análogos & derivados , Carioferinas/metabolismo , Camundongos , MicroRNAs/química , Domínios Proteicos , Capuzes de RNA , Clivagem do RNA , Interferência de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/química , Processamento Pós-Transcricional do RNA , Transporte de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonuclease III/química , Proteína Exportina 1RESUMO
Understanding the structural basis for evolutionary changes in protein function is central to molecular evolutionary biology and can help determine the extent to which functional convergence occurs through similar or different structural mechanisms. Here, we combine ancestral sequence reconstruction with functional characterization and structural modeling to directly examine the evolution of sequence-structure-function across the early differentiation of animal and plant Dicer/DCL proteins, which perform the first molecular step in RNA interference by identifying target RNAs and processing them into short interfering products. We found that ancestral Dicer/DCL proteins evolved similar increases in RNA target affinities as they diverged independently in animal and plant lineages. In both cases, increases in RNA target affinities were associated with sequence changes that anchored the RNA's 5'phosphate, but the structural bases for 5'phosphate recognition were different in animal versus plant lineages. These results highlight how molecular-functional evolutionary convergence can derive from the evolution of unique protein structures implementing similar biochemical mechanisms.
Assuntos
Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Evolução Molecular , Redes Reguladoras de Genes/genética , MicroRNAs/genética , Modelos Genéticos , Filogenia , Proteínas de Plantas/genética , Plantas/genética , RNA de Plantas/genética , Análise de Sequência de Proteína/métodosRESUMO
Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the ß1-ß2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications.
Assuntos
Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA/métodos , Sequência de Aminoácidos/genética , Animais , Evolução Biológica , Bases de Dados de Ácidos Nucleicos , Evolução Molecular , Ligantes , Dados de Sequência Molecular , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Plantas/genética , Ligação Proteica/genética , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Although resurrecting ancestral proteins is a powerful tool for understanding the molecular-functional evolution of gene families, nearly all studies have examined proteins functioning in relatively stable biological processes. The extent to which more dynamic systems obey the same 'rules' governing stable processes is unclear. Here we present the first detailed investigation of the functional evolution of the RIG-like receptors (RLRs), a family of innate immune receptors that detect viral RNA in the cytoplasm. RESULTS: Using kinetic binding assays and molecular dynamics simulations of ancestral proteins, we demonstrate how a small number of adaptive protein-coding changes repeatedly shifted the RNA preference of RLRs throughout animal evolution by reorganizing the shape and electrostatic distribution across the RNA binding pocket, altering the hydrogen bond network between the RLR and its RNA target. In contrast to observations of proteins involved in metabolism and development, we find that RLR-RNA preference 'flip flopped' between two functional states, and shifts in RNA preference were not always coupled to gene duplications or speciation events. We demonstrate at least one reversion of RLR-RNA preference from a derived to an ancestral function through a novel structural mechanism, indicating multiple structural implementations of similar functions. CONCLUSIONS: Our results suggest a model in which frequent shifts in selection pressures imposed by an evolutionary arms race preclude the long-term functional optimization observed in stable biological systems. As a result, the evolutionary dynamics of immune receptors may be less constrained by structural epistasis and historical contingency.
Assuntos
Evolução Molecular , Imunidade Inata , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Sequência de Aminoácidos , Animais , Duplicação Gênica , Humanos , Modelos Moleculares , Filogenia , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
The RIG-like receptors (RLRs) are related proteins that identify viral RNA in the cytoplasm and activate cellular immune responses, primarily through direct protein-protein interactions with the signal transducer, IPS1. Although it has been well established that the RLRs, RIG-I and MDA5, activate IPS1 through binding between the twin caspase activation and recruitment domains (CARDs) on the RLR and a homologous CARD on IPS1, it is less clear which specific RLR CARD(s) are required for this interaction, and almost nothing is known about how the RLR-IPS1 interaction evolved. In contrast to what has been observed in the presence of immune-modulating K63-linked polyubiquitin, here we show that-in the absence of ubiquitin-it is the first CARD domain of human RIG-I and MDA5 (CARD1) that binds directly to IPS1 CARD, and not the second (CARD2). Although the RLRs originated in the earliest animals, both the IPS1 gene and the twin-CARD domain architecture of RIG-I and MDA5 arose much later in the deuterostome lineage, probably through a series of tandem partial-gene duplication events facilitated by tight clustering of RLRs and IPS1 in the ancestral deuterostome genome. Functional differentiation of RIG-I CARD1 and CARD2 appears to have occurred early during this proliferation of RLR and related CARDs, potentially driven by adaptive coevolution between RIG-I CARD domains and IPS1 CARD. However, functional differentiation of MDA5 CARD1 and CARD2 occurred later. These results fit a general model in which duplications of protein-protein interaction domains into novel gene contexts could facilitate the expansion of signaling networks and suggest a potentially important role for functionally-linked gene clusters in generating novel immune-signaling pathways.