comTAR: a web tool for the prediction and characterization of conserved microRNA targets in plants

Motivation: MicroRNAs (miRNAs) are major regulators of gene expression in plants and animals. They recognize their target messenger RNAs (mRNAs) by sequence complementarity and guide them to cleavage or translational arrest. So far, the prediction of plant miRNA–target pairs generally relies on the use of empirical parameters deduced from known miRNA–target interactions. Results: We developed comTAR, a web tool for the prediction of miRNA targets that is mainly based on the conservation of the potential regulation in different species. We used data generated from a pipeline applied to transcript datasets of 33 angiosperms that was used to build a database of potential miRNA targets of different plant species. The database contains information describing each miRNA–target pair, their function and evolutionary conservation, while the results are displayed in a user-friendly interface. The tool also allows the search using new miRNAs.Fil: Chorostecki, Uciel Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Multiple RNA recognition patterns during microRNA biogenesis in plants

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.Fil: Bologna, Nicolas Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Schapire, Arnaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Zhai, Jixian. University of Delaware. Department of Plant & Soil Sciences; Estados Unidos. Delaware Biotechnology Institute; Estados UnidosFil: Chorostecki, Uciel Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Boisbouvier, Jerome. Institut de Biologie Structurale Jean-Pierre Ebel; FranciaFil: Meyers, Blake C.. University of Delaware. Department of Plant & Soil Sciences; Estados Unidos. Delaware Biotechnology Institute; Estados UnidosFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

Identification of key sequence features required for microRNA biogenesis in plants

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis

Estudios sobre la regulación de la expresión génica por microARNs en plantas mediante estrategias bioinformáticas

Los microARNs (o miARNs) son ARN no codificantes que regulan la expresión génica en animales y plantas, y están implicados en procesos biológicos muy variables, como el desarrollo, la diferenciación y el metabolismo. Con un largo de aproximadamente 21 nucleótidos, los miARNs reconocen secuencias parcialmente complementarias en los ARNm blanco, provocando su corte o arresto de la traducción. Los miARNs han saltado rápidamente a la primera plana del interés de la comunidad científica como un nuevo nivel en el control de la expresión génica en eucariotas. Estudios recientes han puesto de manifiesto que los miARNs están implicados en distintas patologías de seres humanos. Los cálculos actuales consideran que cerca del 40% de los genes de humanos se encuentran regulados por miARNs. Está generalmente aceptado que los miARNs en plantas tienen una extensiva complementariedad con sus genes blanco y su predicción por lo general se basa en el uso de parámetros empíricos deducidos de interacciones conocidas y validadas experimentalmente. En este trabajo, primero desarrollamos una estrategia para la identificación de genes blanco regulados por miARNs en plantas, basado en la conservación evolutiva del par miARN-gen blanco. Además, pudimos encontrar genes blanco específicos de Solanaceae y demostrar que la estrategia se puede utilizar para la búsqueda de genes blanco pertenecientes a un grupo determinado de especies. Esta estrategia fue usada para predecir nuevas interacciones no conocidas anteriormente en Arabidopsis thaliana, que luego fueron validados experimentalmente. Algunos de los nuevos genes identificados podrían participar de las mismas vías que genes blanco conocidos anteriormente, sugiriendo que algunos miARNs pueden controlar diferentes aspectos de un proceso biológico. A partir de estos resultados, desarrollamos una herramienta bioinformática disponible en un servidor de acceso público para identificar genes blanco de miARNs basada principalmente en la conservación durante la evolución de la interacción del par miARN-gen blanco en distintas especies. La herramienta también brinda una descripción de varios parámetros de las interacciones miARN-gen blanco en distintas especies, que puede ser útil para mejorar los sistemas de predicción y describir la co-evolucion de los miARNs y los genes blanco. La biogénesis de los miARNs es un proceso clave porque determina la secuencia exacta de nucleótidos del ARN pequeño funcional, que luego determina los genes a ser regulados. Los precursores de miARNs de plantas son muy variables en forma y tamaño en comparación con los precursores de miARNs de plantas. Y por lo tanto, poco se sabía sobre el reconocimiento de los precursores por la maquinaria de procesamiento. En la segunda parte de este trabajo presentamos una estrategia para estudiar la conservación de los precursores de miARNs de plantas en distintas especies identificando regiones y dominios presentes en distintas especies. A partir de estos resultados, desarrollamos un herramienta de visualización para poder analizar fácilmente la conservación de la estructura primaria y secundaria de los precursores de miARNs. El análisis las mismas reveló que existe una marcada correlación entre la conservación de los precursores y el mecanismo de procesamiento. Los resultados muestran patrones de onservación en los dominios necesarios para el procesamiento, y permiten deducir un modelo general del reconocimiento del ARN durante la biogénesis de miARNs.Fil: Chorostecki, Uciel Pablo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Spatial control of gene expression by miR319-regulated TCP transcription factors in leaf development

The characteristic leaf shapes we see in all plants are in good part the outcome of the combined action of several transcription factor networks that translate into cell division activity during the early development of the organ. We show here that wild-type leaves have distinct transcriptomic profiles in center and marginal regions. Certain transcripts are enriched in margins, including those of CINCINNATA-like TCPs (TEOSINTE BRANCHED, CYCLOIDEA and PCF1/2) and members of the NGATHA and STYLISH gene families. We study in detail the contribution of microRNA319 (miR319)-regulated TCP transcription factors to the development of the center and marginal regions of Arabidopsis (Arabidopsis thaliana) leaves. We compare in molecular analyses the wild type, the tcp2 tcp4 mutant that has enlarged flat leaves, and the tcp2 tcp3 tcp4 tcp10 mutant with strongly crinkled leaves. The different leaf domains of the tcp mutants show changed expression patterns for many photosynthesisrelated genes, indicating delayed differentiation, especially in the marginal parts of the organ. At the same time, we found an up-regulation of cyclin genes and other genes that are known to participate in cell division, specifically in the marginal regions of tcp2 tcp3 tcp4 tcp10. Using GUS reporter constructs, we confirmed extended mitotic activity in the tcp2 tcp3 tcp4 tcp10 leaf, which persisted in small defined foci in the margins when the mitotic activity had already ceased in wild-type leaves. Our results describe the role of miR319-regulated TCP transcription factors in the coordination of activities in different leaf domains during organ development.Fil: Bresso, Edgardo G.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario, Argentina; ArgentinaFil: Chorostecki, Uciel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Rodriguez, Ramiro E.. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Schommer, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario; Argentin

Efficiency and precision of microRNA biogenesis modes in plants

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 wildtype 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.This article has been accepted for publication in Nucleic Acids Research Published by Oxford University Press.Fil: Moro, Belén. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Moro, Belén. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Chorostecki, Uciel Pablo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Chorostecki, Uciel Pablo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Arikit, Siwaret. Kasetsart University. Department of Agronomy, Kamphaeng Saen and Rice Science Center; Thailand.Fil: Suárez, Irina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Höbartner, Claudia. Universität Würzburg. Institut für Organische Chemie; Deutchland.Fil: Rasia, Rodolfo M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Rasia, Rodolfo M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Meyers, Blake C. Donald Danforth Plant Science Center; United States.Fil: Meyers, Blake C. University of Missouri. Department of Plant Science; United States.Fil: Palatnik, Javier F. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Palatnik, Javier F. Universidad Nacional de Rosario. Centro de Estudios Interdisciplinarios (CEI); Argentina

Secondary structure prediction of long noncoding RNA: review and experimental comparison of existing approaches

MOTIVATION: In contrast to messenger RNAs, the function of the wide range of existing long noncoding RNAs (lncRNAs) largely depends on their structure, which determines interactions with partner molecules. Thus, the determination or prediction of the secondary structure of lncRNAs is critical to uncover their function. Classical approaches for predicting RNA secondary structure have been based on dynamic programming and thermodynamic calculations. In the last 4 years, a growing number of machine learning (ML)-based models, including deep learning (DL), have achieved breakthrough performance in structure prediction of biomolecules such as proteins and have outperformed classical methods in short transcripts folding. Nevertheless, the accurate prediction for lncRNA still remains far from being effectively solved. Notably, the myriad of new proposals has not been systematically and experimentally evaluated. RESULTS: In this work, we compare the performance of the classical methods as well as the most recently proposed approaches for secondary structure prediction of RNA sequences using a unified and consistent experimental setup. We use the publicly available structural profiles for 3023 yeast RNA sequences, and a novel benchmark of well-characterized lncRNA structures from different species. Moreover, we propose a novel metric to assess the predictive performance of methods, exclusively based on the chemical probing data commonly used for profiling RNA structures, avoiding any potential bias incorporated by computational predictions when using dot-bracket references. Our results provide a comprehensive comparative assessment of existing methodologies, and a novel and public benchmark resource to aid in the development and comparison of future approaches.This work was supported by ANPCyT (PICT 2018 3384, PICT 2018 2905, PICT 2019 3420) and UNL (CAI+D 2020 115). Researchers from sinc(i) and IAL are collaborating in the framework of the Program Science and Technology against Hunger, supported by the Argentinian Ministry of Science, to study and develop ncRNAs as exogenous bioactive molecules in agriculture. UC was funded by MICINN (IJC2019-039402-I). The work used computational resources from the Pirayu cluster, acquired with funds from the Santa Fe Science, Technology and Innovation Agency (ASACTEI), Project AC-00010-18, Res. No. 117/14. This equipment is part of the National High Performance Computing System of the Ministry of Science and Technology of Argentina. We also acknowledged the support of NVIDIA Corporation for the donation of GPUs used for this researchPeer Reviewed"Article signat per 12 autors/es: L A Bugnon, A A Edera, S Prochetto, M Gerard, J Raad, E Fenoy, M Rubiolo, U Chorostecki, T Gabaldón, F Ariel, L E Di Persia, D H Milone, G Stegmayer"Postprint (author's final draft

Evolutionary footprints reveal insights into plant microRNA biogenesis

MicroRNAs (miRNAs) are endogenous small RNAs that recognize target sequences by base complementarity and play a role in the regulation of target gene expression. They are processed from longer precursor molecules that harbor a fold-back structure. Plant miRNA precursors are quite variable in size and shape, and are recognized by the processing machinery in different ways. However, ancient miRNAs and their binding sites in target genes are conserved during evolution. Here, we designed a strategy to systematically analyze MIRNAs from different species generating a graphical representation of the conservation of the primary sequence and secondary structure. We found that plant MIRNAs have evolutionary footprints that go beyond the small RNA sequence itself, yet their location along the precursor depends on the specific MIRNA. We show that these conserved regions correspond to structural determinants recognized during the biogenesis of plant miRNAs. Furthermore, we found that the members of the miR166 family have unusual conservation patterns and demonstrated that the recognition of these precursors in vivo differs from other known miRNAs. Our results describe a link between the evolutionary conservation of plant MIRNAs and the mechanisms underlying the biogenesis of these small RNAs and show that the MIRNA pattern of conservation can be used to infer the mode of miRNA biogenesis.Fil: Chorostecki, Uciel Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moro, Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rojas, Arantxa Maria Larisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Debernardi, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Schapire, Arnaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Notredame, Cedric. The Barcelona Institute of Science and Technology; España. Universitat Pompeu Fabra; EspañaFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Centro de Estudios Interdisciplinarios; Argentin

Identification of key sequence features required for microRNA biogenesis in plants

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis