ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , MicroRNAs/genetics , Phosphoric Monoester Hydrolases/genetics , RNA-Binding Proteins/genetics , Binding Sites , Computational Biology , Gene Expression Regulation, Plant , Gene Library , MicroRNAs/biosynthesis , Mutation , Plants, Genetically Modified , Polymerase Chain Reaction , Protein Structure, Secondary , RNA Processing, Post-Transcriptional , RNA, Double-Stranded/genetics , Seedlings , Transcription, Genetic , TransgenesABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , MicroRNAs/metabolism , Ribonuclease III/metabolism , Arabidopsis , Arabidopsis Proteins/chemistry , Cell Cycle Proteins/chemistry , Circular Dichroism , Magnetic Resonance Spectroscopy , MicroRNAs/chemistry , Models, Chemical , Protein Binding , Protein Conformation , Protein Domains , Protein Folding , Ribonuclease III/chemistry , Scattering, Small Angle , Temperature , X-Ray DiffractionABSTRACT
The intrinsically disordered protein domain DCL1-A is the first report of a complete double stranded RNA binding domain folding upon binding. DCL1-A recognizes the dsRNA by acquiring a well-folded structure after engagement with its interaction partner. Despite the structural characterization of the interaction complex underlying the recognition of dsRNA has been established, the dynamics of disorder-to-order transitions in the binding process remains elusive. Here we have developed a coarse-grained structure-based model with consideration of electrostatic interactions to explore the mechanism of the coupled folding and binding. Our approach led to remarkable agreements with both experimental and theoretical results. We quantified the global binding-folding landscape, which indicates a synergistic binding induced folding mechanism. We further investigated the effect of electrostatic interactions in this coupled folding and binding process. It reveals that non-native electrostatic interactions dominate the initial stage of the recognition. Our results help improve our understanding of the induced folding of the IDP DCL1-A upon binding to dsRNA. Such methods developed here can be applied for further explorations of the dynamics of coupled folding and binding systems.
Subject(s)
Arabidopsis Proteins/chemistry , Cell Cycle Proteins/chemistry , Protein Folding , RNA/chemistry , Ribonuclease III/chemistry , Computer Simulation , Kinetics , Models, Molecular , Protein Binding , Protein Domains , Static Electricity , Structure-Activity Relationship , ThermodynamicsABSTRACT
Double stranded RNA (dsRNA) participates in several biological processes, where RNA molecules acquire secondary structure inside the cell through base complementarity. The double stranded RNA binding domain (dsRBD) is one of the main protein folds that is able to recognize and bind to dsRNA regions. The N-terminal dsRBD of DCL1 in Arabidopsis thaliana (DCL1-1), in contrast to other studied dsRBDs, lacks a stable structure, behaving as an intrinsically disordered protein. DCL1-1 does however recognize dsRNA by acquiring a canonical fold in the presence of its substrate. Here we present a detailed modeling and molecular dynamics study of dsRNA recognition by DCL1-1. We found that DCL1-1 forms stable complexes with different RNAs and we characterized the residues involved in binding. Although the domain shows a binding loop substantially shorter than other homologs, it can still interact with the dsRNA and results in bending of the dsRNA A-type helix. Furthermore, we found that R8, a non-conserved residue located in the first dsRNA binding region, recognizes preferentially mismatched base pairs. We discuss our findings in the context of the function of DCL1-1 within the microRNA processing complex.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , Cell Cycle Proteins/chemistry , MicroRNAs/chemistry , Models, Chemical , Molecular Dynamics Simulation , RNA, Double-Stranded/chemistry , RNA, Plant/chemistry , Ribonuclease III/chemistry , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , MicroRNAs/metabolism , RNA, Double-Stranded/metabolism , RNA, Plant/metabolism , Ribonuclease III/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/chemistry , Cell Cycle Proteins/chemistry , RNA-Binding Proteins/chemistry , Ribonuclease III/chemistry , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Models, Molecular , Protein Binding , Protein Stability , Protein Structure, Tertiary , RNA/chemistry , RNA/metabolism , RNA Folding , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolismABSTRACT
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.
