Keeping up with the miRNAs: current paradigms of the biogenesis pathway.

For many years we have studied the processes involved in producing miRNAs in plants and the numerous differences from their metazoan counterpart. A well-defined catalytic process, mostly carried out by the RNase III enzyme DICER-LIKE1 (DCL1), it was identified early after the discovery of RNAi and was followed by the isolation of a plethora of miRNA biogenesis cofactors. The production of miRNAs, which later are loaded in ARGONAUTE (AGO) proteins to perform their RNA silencing functions both within the cell and non-cell autonomously, appears to be a highly regulated and dynamic process. Many regulatory events during miRNA biogenesis require the action of specific proteins. However, in recent years, many post-transcriptional modifications, structural features, and coupling with other cellular processing emerged as critical elements controlling the production of miRNA and, thus, a plant's physiology. This review discusses new evidence that has changed the way we understand how miRNAs are produced in plants. We also provide an updated view of the miRNA biogenesis pathways, focusing on the gaps in our knowledge and the most compelling questions that remain open.

Genome-wide identification of the Dicer-like family in cotton and analysis of the DCL expression modulation in response to biotic stress in two contrasting commercial cultivars.

BACKGROUND: Dicer-like proteins (DCLs) are essential players in RNA-silencing mechanisms, acting in gene regulation via miRNAs and in antiviral protection in plants and have also been associated to other biotic and abiotic stresses. To the best of our knowledge, despite being identified in some crops, cotton DCLs haven't been characterized until now. In this work, we characterized the DCLs of three cotton species and analyzed their expression profiles during biotic stress. RESULTS: As main results, 11 DCLs in the allotetraploid cotton Gossypium hirsutum, 7 and 6 in the diploid G. arboreum and G. raimondii, were identified, respectively. Among some DCLs duplications observed in these genomes, the presence of an extra DCL3 in the three cotton species were detected, which haven't been found in others eudicots. All the DCL types identified by in silico analysis in the allotetraploid cotton genome were able to generate transcripts, as observed by gene expression analysis in distinct tissues. Based on the importance of DCLs for plant defense against virus, responses of cotton DCLs to virus infection and/or herbivore attack using two commercial cotton cultivars (cv.), one susceptible (FM966) and another resistant (DO) to polerovirus CLRDV infection, were analyzed. Both cvs. Responded differently to virus infection. At the inoculation site, the resistant cv. showed strong induction of DCL2a and b, while the susceptible cv. showed a down-regulation of these genes, wherever DCL4 expression was highly induced. A time course of DCL expression in aerial parts far from inoculation site along infection showed that DCL2b and DCL4 were repressed 24 h after infection in the susceptible cotton. As CLRDV is aphid-transmitted, herbivore attack was also checked. Opposite expression pattern of DCL2a and b and DCL4 was observed for R and S cottons, showing that aphid feeding alone may induce DCL modulation. CONCLUSIONS: Almost all the DCLs of the allotetraploide G. hirsutum cotton were found in their relative diploids. Duplications of DCL2 and DCL3 were found in the three species. All four classes of DCL responded to aphid attack and virus infection in G. hirsutum. DCLs initial responses against the virus itself and/or herbivore attack may be contributing towards virus resistance.

Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis.

BACKGROUND: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. RESULTS: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. CONCLUSIONS: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Arabidopsis thaliana miRNAs promote embryo pattern formation beginning in the zygote.

miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.

Genome-wide identification and in silico characterisation of microRNAs, their targets and processing pathway genes in Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L., Fabaceae) is a globally important staple crop, which is an important source of calories, protein and essential micronutrients. At the genomic level little is known regarding the small non-coding RNAs within the common bean genome. One of the most important classes of such small non-coding RNAs is microRNAs (miRNAs), which control mRNA and protein expression levels in many eukaryotes. Computational methods have been applied to identify putative miRNAs in the genomes of different organisms. In this study, our objective was to comprehensively identify and characterise miRNAs from the genome and transcriptome of P. vulgaris, including both mature and precursor miRNA forms. We also sought to identify the putative proteins involved in miRNA processing and the likely target genes of common bean miRNAs. We identified 221 mature miRNAs and 136 precursor miRNAs distributed across 52 different miRNA families in the P. vulgaris genome. Amongst these, we distinguished 129 novel mature miRNAs and 123 miRNA precursors belonging to 24 different miRNA families. We also identified 31 proteins predicted to participate in the miRNA-processing pathway in P. vulgaris. Finally, we also identified 483 predicted miRNA targets, including many which corroborate results from other species, suggesting that miRNA regulatory systems are evolutionarily conserved and important for plant development. Our results expand the study of miRNAs and their target genes in common bean, and provide new opportunities to understand their roles in the biology of this important staple crop.

Evolución de la categorización semántica en adultos mayores con diagnóstico de DCL-A y DTA y sin patología neurológica / Evolution of semantic categorization in older adults with and without neurological disease

Tradicionalmente la memoria semántica ha sido definida como un tesoro mental que almacena información sobre las palabras, su significado, como así también la relación entre ellas, los hechos y los conceptos. Una de las vías principales para explicar cómo se organiza la información en la memoria semántica (MS) es a través de categorías. La demencia tipo Alzheimer (DTA) suele estar caracterizada por un deterioro en la memoria episódica. Sin embargo, se debate en torno al deterioro de la misma, en qué momento aparece, a qué se debe y si hay una pérdida diferencial en distintos momentos de la enfermedad. El objetivo del trabajo que se informa fue analizar los procesos de categorización semántica en adultos mayores sanos comparándolos con dos grupos de personas con patologías neurológicas: uno con diagnóstico de deterioro cognitivo leve (DCL) y otro con demencia tipo Alzheimer (DTA), emparejados por edad, nivel educativo y género. Para ello, se utilizaron tareas tradicionales para evaluar la memoria semántica y también un método novedoso, el DISTSEM, que permite evaluar las estimaciones que realizan las personas de las distancias semánticas entre los conceptos. Los resultados mostraron que el grupo de personas sin patología tuvo un rendimiento superior en todas las tareas en comparación con los grupos de personas con patología neurológica. Por otro lado, el grupo con DTA mostró un deterioro semántico afectando en primer lugar a la categoría seres vivos. El DCL preservó la estructura categorial sólo con pequeñas intrusiones. El DISTSEM mostró ser un instrumento adecuado para la discriminación entre sujetos sanos y DTA.

Traditionally, semantic memory has been defined as a mental trove that contains organized information about words and their meaning, as well as relationships between words, facts and concepts.There are different ways of explaining how the information in such memory system is represented and organized. One of the main ways of explaining this organization is through categories. Categorization starts the very moment that any sensorial perception is associated to an abstract category; it is the ability to organize information in equivalence classes, and it is very important because it allows us to summarize the information that we gather through our senses and thus facilitate its manipulation. Alzheimer's disease (AD) is the main cause of dementia among older adults, and it is one of the most pressing sanitary, social and cultural problems of the present times. The main risk factor for the illness is age, for its prevalence augments exponentially from 65 to 85 years old, and due to the rise in life expectancy it is considered that it might be transformed into a world-wide epidemic in the coming years. The start of the illness is insidious and slowly progressing, and it is characterized by a loss of episodic memory from its beginnings. With respect to the deterioration of semantic memory in AD, a debate has arisen about its mode of presentation, its causes, and whether there is a differential loss of categories (live or non-live beings) in different moments of the illness. During the 90s, Petersen proposed the concept of mild cognitive impairment (MCI) to label the subjects with a functional cognitive impairment insufficient to diagnose a dementia syndrome. The importance of this syndrome is that between 8 to 15% of those who suffer it evolve to AD annually, while in the general population this evolution is only from 1 to 2%. However, there is a great controversy about the validity and scope of the term MCI, for its etiology can be very varied and it is very difficult to predict its evolution. There are different sub-kinds of MCI, and the amnesic form is the more likely to evolve into AD, for its main characteristic is the subjective deterioration of memory corroborated by standardized test by reference to normative data for the same age and educational level of the subject. The other cognitive functions not present alterations. The main objective of this work was to analyze the semantic categorization processes in healthy older adults comparing them with two groups of people with neurological pathologies, one with diagnosis of MCI and the other with AD, matched by age, educational level and gender. To evaluate this categorization process, two traditional tasks were used together with a novel method, the DISTSEM, which allows the evaluation of the estimation made by people about the semantic distance among a set of given concepts. The results showed that the group of people without pathologies had a superior performance in all the tasks in comparison with the groups of people with neurological pathologies. On the other hand, the group with AD showed a semantic impairment which affected in the first place the category of live beings, which was evident in the all the tasks. The MCI preserved the categorical structure with only minor intrusions. The DISTSEM has shown itself to be a valuable instrument that allows the discrimination between healthy subjects and those with AD. It is expected that this work can provide empirical evidence relevant to the debate surrounding semantic memory, for studies in patients with brain injury offer key information to examine the organizing models of such memory.

Evolución de la categorización semántica en adultos mayores con diagnóstico de DCL-A y DTA y sin patología neurológica / Evolution of semantic categorization in older adults with and without neurological disease

Tradicionalmente la memoria semántica ha sido definida como un tesoro mental que almacena información sobre las palabras, su significado, como así también la relación entre ellas, los hechos y los conceptos. Una de las vías principales para explicar cómo se organiza la información en la memoria semántica (MS) es a través de categorías. La demencia tipo Alzheimer (DTA) suele estar caracterizada por un deterioro en la memoria episódica. Sin embargo, se debate en torno al deterioro de la misma, en qué momento aparece, a qué se debe y si hay una pérdida diferencial en distintos momentos de la enfermedad. El objetivo del trabajo que se informa fue analizar los procesos de categorización semántica en adultos mayores sanos comparándolos con dos grupos de personas con patologías neurológicas: uno con diagnóstico de deterioro cognitivo leve (DCL) y otro con demencia tipo Alzheimer (DTA), emparejados por edad, nivel educativo y género. Para ello, se utilizaron tareas tradicionales para evaluar la memoria semántica y también un método novedoso, el DISTSEM, que permite evaluar las estimaciones que realizan las personas de las distancias semánticas entre los conceptos. Los resultados mostraron que el grupo de personas sin patología tuvo un rendimiento superior en todas las tareas en comparación con los grupos de personas con patología neurológica. Por otro lado, el grupo con DTA mostró un deterioro semántico afectando en primer lugar a la categoría seres vivos. El DCL preservó la estructura categorial sólo con pequeñas intrusiones. El DISTSEM mostró ser un instrumento adecuado para la discriminación entre sujetos sanos y DTA.(AU)

Traditionally, semantic memory has been defined as a mental trove that contains organized information about words and their meaning, as well as relationships between words, facts and concepts.There are different ways of explaining how the information in such memory system is represented and organized. One of the main ways of explaining this organization is through categories. Categorization starts the very moment that any sensorial perception is associated to an abstract category; it is the ability to organize information in equivalence classes, and it is very important because it allows us to summarize the information that we gather through our senses and thus facilitate its manipulation. Alzheimers disease (AD) is the main cause of dementia among older adults, and it is one of the most pressing sanitary, social and cultural problems of the present times. The main risk factor for the illness is age, for its prevalence augments exponentially from 65 to 85 years old, and due to the rise in life expectancy it is considered that it might be transformed into a world-wide epidemic in the coming years. The start of the illness is insidious and slowly progressing, and it is characterized by a loss of episodic memory from its beginnings. With respect to the deterioration of semantic memory in AD, a debate has arisen about its mode of presentation, its causes, and whether there is a differential loss of categories (live or non-live beings) in different moments of the illness. During the 90s, Petersen proposed the concept of mild cognitive impairment (MCI) to label the subjects with a functional cognitive impairment insufficient to diagnose a dementia syndrome. The importance of this syndrome is that between 8 to 15% of those who suffer it evolve to AD annually, while in the general population this evolution is only from 1 to 2%. However, there is a great controversy about the validity and scope of the term MCI, for its etiology can be very varied and it is very difficult to predict its evolution. There are different sub-kinds of MCI, and the amnesic form is the more likely to evolve into AD, for its main characteristic is the subjective deterioration of memory corroborated by standardized test by reference to normative data for the same age and educational level of the subject. The other cognitive functions not present alterations. The main objective of this work was to analyze the semantic categorization processes in healthy older adults comparing them with two groups of people with neurological pathologies, one with diagnosis of MCI and the other with AD, matched by age, educational level and gender. To evaluate this categorization process, two traditional tasks were used together with a novel method, the DISTSEM, which allows the evaluation of the estimation made by people about the semantic distance among a set of given concepts. The results showed that the group of people without pathologies had a superior performance in all the tasks in comparison with the groups of people with neurological pathologies. On the other hand, the group with AD showed a semantic impairment which affected in the first place the category of live beings, which was evident in the all the tasks. The MCI preserved the categorical structure with only minor intrusions. The DISTSEM has shown itself to be a valuable instrument that allows the discrimination between healthy subjects and those with AD. It is expected that this work can provide empirical evidence relevant to the debate surrounding semantic memory, for studies in patients with brain injury offer key information to examine the organizing models of such memory.(AU)

Puntuaciones del MoCA y el MMSE en pacientes con deterioro cognitivo leve y demencia en una clínica de memoria en Bogotá / MoCA and MMSE scores in patients with mild cognitive impairment and dementia in a memory clinic in Bogotá

Introducción. Diferentes pruebas neuropsicológicas permiten explorar las funciones cognitivas del adulto mayor, en un tiempo corto. En Colombia se dispone de pocos estudios sobre puntuaciones y puntos de corte para el MMSE y para el MoCA en relación al diagnóstico de deterioro cognitivo. Objetivo. Describir la distribución de las puntuaciones del MMSE y el MoCA y los puntos de corte con mejor discriminación, para el diagnóstico de deterioro cognitivo leve y demencia, en una muestra de pacientes de Bogotá. Material y métodos. Se evaluaron 248 pacientes por un equipo multidisciplinario, que consultaron a la Clínica de Memoria del HIUSJ entre 2009-2012, siguiendo un protocolo establecido. Se identificaron las puntuaciones del MoCA y MMSE, que permitieron obtener el mayor porcentaje de pacientes correctamente clasificados. Resultados. En el 70% de los pacientes con DCL y en el 69 % de los sujetos normales, se encontraron puntuaciones del MMSE inferiores o iguales a 28. En 91% de pacientes con DCL y 84% de los sujetos normales, se presentaron puntuaciones del MoCA inferiores o iguales a 25. Los pacientes con cualquier tipo de demencia, presentaron puntuaciones del MMSE inferiores o iguales a 27 e inferiores o iguales a 24 en el MoCA. Conclusión. Según el presente estudio, el tamizaje de funciones cognitivas, utilizando el MoCA, clasifica de manera más acertada que el MMSE, a los sujetos con deterioro cognitivo. Creemos que en atención primaria, estos puntos de corte del MoCA, pueden ser considerados por ahora, cuando se trate especialmente de sujetos con alta escolaridad.

Introduction. Some cognitive tests allow the evaluation of cognitive functions on the elderly in a short period of time. There are few studies in Colombia about cut-off point for the MMSE and the MoCA test. Objectives. To describe the distribution on scores on MMSE and MoCA test and the cut-off point with a better discrimination criteria for the diagnosis of mild cognitive impairment and dementia, in a sample of patients from Bogotá. Materials and methods. Two hundred forty eight patients were included in this study, being evaluated by a multidisciplinary team that followed an established protocol, on patients who attended to the Memory Clinic of HIUSJ between 2009-2012. MoCA test and MMSE scores that allow higher percentages of correctly classified patients were identified. Results. Seventy percent of patients with mild cognitive impairment and 69% of normal individuals had scores on MMSE below or equal to 28. Ninety-one percent of patients with MCI and 89% of normal patients, had scores below or equal to 25. Patients with any type of dementia had scores on MMSE below or equal to 27 and below or equal to 24 in MoCA test. Conclusion. According to the study, the screening of cognitive functions, using MoCA test, is more accurate than MMSE in patients with cognitive decline. The cut-off points, identified in our study, can be considered useful until now in primary attention, in patients with a high level of education.

Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development.

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.