RESUMO
MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.
Assuntos
MicroRNAs , Processamento Pós-Transcricional do RNA , RNA de Plantas , Ribonuclease III , MicroRNAs/genética , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/química , Ribonuclease III/metabolismo , Ribonuclease III/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Conformação de Ácido Nucleico , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo CelularRESUMO
Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , MicroRNAs/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas de Ligação a RNA/genética , Sítios de Ligação , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , MicroRNAs/biossíntese , Mutação , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Processamento Pós-Transcricional do RNA , RNA de Cadeia Dupla/genética , Plântula , Transcrição Gênica , TransgenesRESUMO
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. Substrate pri-miRNA recognition by DCL1 requires two double stranded RNA binding domains located at the C-terminus of the protein. We have previously shown that the first of these domains, DCL1-A, is intrinsically disordered and folds upon binding pri-miRNA. Integrating NMR and SAXS data, we study here the conformational landscape of free DCL1-A through an ensemble description. Our results reveal that secondary structure elements, corresponding to the folded form of the protein, are transiently populated in the unbound state. The conformation of one of the dsRNA binding regions in the free protein shows that, at a local level, RNA recognition proceeds through a conformational selection mechanism. We further explored the stability of the preformed structural elements via temperature and urea destabilization. The C-terminal helix is halfway on the folding pathway in free DCL1-A, constituting a potential nucleation site for the final folding of the protein. In contrast, the N-terminal helix adopts stable non-native structures that could hinder the correct folding of the protein in the absence of RNA. This description of the unfolded form allows us to understand details of the mechanism of binding-induced folding of the protein.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/química , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , MicroRNAs/química , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Ribonuclease III/química , Espalhamento a Baixo Ângulo , Temperatura , Difração de Raios XRESUMO
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Dobramento de RNA , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismoRESUMO
Dicer-like ribonuclease III enzymes are involved in different paths related to RNA silencing in plants. Little is known about the structural aspects of these processes. Here we present a structural characterization of the second double-stranded RNA binding domain (dsRBD) of DCL1, which is presumed to participate in pri-micro-RNA recognition and subcellular localization of this protein. We determined the solution structure and found that it has a canonical fold but bears some variation with respect to other homologous domains. We also found that this domain binds both double-stranded RNA and double-stranded DNA, in contrast to most dsRBDs. Our characterization shows that this domain likely has functions other than substrate recognition and binding.