LIPRNAseq: a method to discover lipid interacting RNAs by sequencing.

BACKGROUND: Current biological research extensively describes the interactions of molecules such as RNA with other nucleic acids or proteins. However, the relatively recent discovery of nuclear phospholipids playing biologically relevant processes outside membranes, as well as, RNA-lipid interactions shows the need for new methods to explore the identity of these RNAs. METHODS AND RESULTS: In this study, we describe the method for LIPID-RNA isolation followed by sequencing and analysis of the RNA that has the ability to interact with the selected lipids. Here we utilized specific phospholipid coated beads for selective RNA binding. We tested RNA from organisms belonging to different realms (human, plant, and yeast), and tested their ability to bind a specific lipid. CONCLUSIONS: The results show several RNAs differentially enriched in the pull-down of phosphatidyl Inositol 4,5 bisphosphate coated beads. This method is helpful to screen lipid-binding RNA, which may have relevant biological functions. The method can be used with different lipids and comparison of pull-downs and can narrow the selection of RNAs that interact with a particular lipid for further studies.

Current research on viral proteins that interact with fibrillarin.

The nucleolus is a multifunctional nuclear domain primarily dedicated to ribosome biogenesis. Certain viruses developed strategies to manipulate host nucleolar proteins to facilitate their replication by modulating ribosomal RNA (rRNA) processing. This association interferes with nucleolar functions resulting in overactivation or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and affecting stress response. The nucleolar protein fibrillarin (FBL) is an important target of some plant and animal viruses. FBL is an essential and highly conserved S-adenosyl methionine (SAM) dependent methyltransferase, capable of rRNA degradation by its intrinsically disordered region (IDR), the glycine/arginine-rich (GAR) domain. It forms a ribonucleoprotein complex that directs 2'-O-methylations in more than 100 sites of pre-rRNAs. It is involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. The interaction with animal viruses, including human viruses, triggered its redistribution to the nucleoplasm and cytoplasm, interfering with its role in pre-rRNA processing. Viral-encoded proteins with IDRs as nucleocapsids, matrix, Tat protein, and even a viral snoRNA, can associate with FBL, forcing the nucleolar protein to undergo atypical functions. Here we review the molecular mechanisms employed by animal and human viruses to usurp FBL functions and the effect on cellular processes, particularly in ribosome biogenesis.

Evaluation of Natural Pigments Production in Response to Various Stress Signals in Cell Lines of Stenocereus queretaroensis.

Stenocereus queretaroensis (F.A.C. Weber ex Mathes.) Buxb is a cactus that has long been used as a source food in central and northern México. Its fruits, commonly called pitayas, biosynthesize high amounts of betalains. These molecules are water-soluble nitrogenous compounds; that compared to other pigments, such as anthocyanins or carotenoids, stand out for their physicochemical stability in industrial processes. Due to genetic and environmental factors involved in the biosynthesis and accumulation of secondary metabolites in plants, we tested different stress-inducing agents (elicitor, osmotic, salt, and temperature) to induce betalains accumulation in cell culture from fruits of Stenocereus queretaroensis. This work aimed to understand stress conditions that induce the metabolic pathways required for the accumulation of betalains. The results show how betacyanin concentration increases under high sugar conditions, thus affecting the expression of L-DOPA 4, 5 dioxygenase resulting in a strong dark red coloration. This suggests this enzyme is part of a rate-limiting step in betalain production. In addition, we found that betalains accumulation occurs under particular stress conditions. Cells that have a high level of betacyanins show better resistance to stress in the cell culture, as well as an overall different behavior including cell aggregation and alterations in nuclear size. Together the results shown here may provide new strategies to manipulate and mass produce the pigments from Stenocereus queretaroensis in cell culture.

Phase Separation of Intrinsically Disordered Nucleolar Proteins Relate to Localization and Function.

The process of phase separation allows for the establishment and formation of subcompartmentalized structures, thus enabling cells to perform simultaneous processes with precise organization and low energy requirements. Chemical modifications of proteins, RNA, and lipids alter the molecular environment facilitating enzymatic reactions at higher concentrations in particular regions of the cell. In this review, we discuss the nucleolus as an example of the establishment, dynamics, and maintenance of a membraneless organelle with a high level of organization.

Plant viral proteins and fibrillarin: the link to complete the infective cycle.

The interaction between viruses with the nucleolus is already a well-defined field of study in plant virology. This interaction is not restricted to those viruses that replicate in the nucleus, in fact, RNA viruses that replicate exclusively in the cytoplasm express proteins that localize in the nucleolus. Some positive single stranded RNA viruses from animals and plants have been reported to interact with the main nucleolar protein, Fibrillarin. Among nucleolar proteins, Fibrillarin is an essential protein that has been conserved in sequence and function throughout evolution. Fibrillarin is a methyltransferase protein with more than 100 methylation sites in the pre-ribosomal RNA, involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. Recently, it was found that AtFib2 shows a ribonuclease activity. In plant viruses, Fibrillarin is involved in long-distance movement and cell-to-cell movement, being two highly different processes. The mechanism that Fibrillarin performs is still unknown. However, and despite belonging to very different viral families, the majority comply with the following. (1) They are positive single stranded RNA viruses; (2) encode different types of viral proteins that partially localize in the nucleolus; (3) interacts with Fibrillarin exporting it to the cytoplasm; (4) the viral protein-Fibrillarin interaction forms an RNP complex with the viral RNA and; (5) Fibrillarin depletion affects the infective cycle of the virus. Here we review the relationship of those plant viruses with Fibrillarin interaction, with special focus on the molecular processes of the virus to sequester Fibrillarin to complete its infective cycle.

Towards an understanding of the role of intrinsic protein disorder on plant adaptation to environmental challenges.

Intrinsic protein disorder is an interesting structural feature where fully functional proteins lack a three-dimensional structure in solution. In this work, we estimated the relative content of intrinsic protein disorder in 96 plant proteomes including monocots and eudicots. In this analysis, we found variation in the relative abundance of intrinsic protein disorder among these major clades; the relative level of disorder is higher in monocots than eudicots. In turn, there is an inverse relationship between the degree of intrinsic protein disorder and protein length, with smaller proteins being more disordered. The relative abundance of amino acids depends on intrinsic disorder and also varies among clades. Within the nucleus, intrinsically disordered proteins are more abundant than ordered proteins. Intrinsically disordered proteins are specialized in regulatory functions, nucleic acid binding, RNA processing, and in response to environmental stimuli. The implications of this on plants' responses to their environment are discussed.

Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids.

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.

The effect of plant stress on phosphoinositides.

Phosphoinositides are very versatile molecules with a plethora of functions such as cytokinesis, chemotaxis, cell survival, and cell death. Their functions depend on the proteins with which they interact. Thus, when interacting with phospholipases, phosphatases, or kinases, they can be precursors of second messengers in different signalling pathways. They could be second messengers themselves and interact directly with other proteins to modulate their functions trough changing its localization and activity or enhancing its synthesis rate. Because they are more abundant in animal cells and their importance in diseases such as cancer has taken priority, the study of the phosphoinositides in plants has not evolved to the same extent. Nevertheless, several studies have shown the significance of these lipids in plant cells viability and environmental response. This review focuses on phosphoinositides response to abiotic and biotic stress, showing their implication in plant survival during different stages of development. SIGNIFICANCE OF THE STUDY: This review is focused on plant PIPs functions in stress, highlighting in the main differences between plant and mammal PIPs and the novel interactions that could be extrapolated to animal models to contribute in a better understanding of these pivotal molecules.

Global Dynamics in Protein Disorder during Maize Seed Development.

Intrinsic protein disorder is a physicochemical attribute of some proteins lacking tridimensional structure and is collectively known as intrinsically disordered proteins (IDPs). Interestingly, several IDPs have been associated with protective functions in plants and with their response to external stimuli. To correlate the modulation of the IDPs content with the developmental progression in seed, we describe the expression of transcripts according to the disorder content of the proteins that they codify during seed development, from the early embryogenesis to the beginning of the desiccation tolerance acquisition stage. We found that the total expression profile of transcripts encoding for structured proteins is highly increased during middle phase. However, the relative content of protein disorder is increased as seed development progresses. We identified several intrinsically disordered transcription factors that seem to play important roles throughout seed development. On the other hand, we detected a gene cluster encoding for IDPs at the end of the late phase, which coincides with the beginning of the acquisition of desiccation tolerance. In conclusion, the expression pattern of IDPs is highly dependent on the developmental stage, and there is a general reduction in the expression of transcripts encoding for structured proteins as seed development progresses. We proposed maize seeds as a model to study the regulation of protein disorder in plant development and its involvement in the acquisition of desiccation tolerance in plants.

Transcriptomics and co-expression networks reveal tissue-specific responses and regulatory hubs under mild and severe drought in papaya (Carica papaya L.).

Plants respond to drought stress through the ABA dependent and independent pathways, which in turn modulate transcriptional regulatory hubs. Here, we employed Illumina RNA-Seq to analyze a total of 18 cDNA libraries from leaves, sap, and roots of papaya plants under drought stress. Reference and de novo transcriptomic analyses identified 8,549 and 6,089 drought-responsive genes and unigenes, respectively. Core sets of 6 and 34 genes were simultaneously up- or down-regulated, respectively, in all stressed samples. Moreover, GO enrichment analysis revealed that under moderate drought stress, processes related to cell cycle and DNA repair were up-regulated in leaves and sap; while responses to abiotic stress, hormone signaling, sucrose metabolism, and suberin biosynthesis were up-regulated in roots. Under severe drought stress, biological processes related to abiotic stress, hormone signaling, and oxidation-reduction were up-regulated in all tissues. Moreover, similar biological processes were commonly down-regulated in all stressed samples. Furthermore, co-expression network analysis revealed three and eight transcriptionally regulated modules in leaves and roots, respectively. Seventeen stress-related TFs were identified, potentially serving as main regulatory hubs in leaves and roots. Our findings provide insight into the molecular responses of papaya plant to drought, which could contribute to the improvement of this important tropical crop.

Does water stress promote the proteome-wide adjustment of intrinsically disordered proteins in plants?

Plant response to water stress involves the activation of mechanisms expected to help them cope with water scarcity. Among these mechanisms, proteome-wide adjustment is well known. This includes actions to save energy, protect cellular and molecular components, and maintain vital functions of the cell. Intrinsically disordered proteins, which are proteins without a rigid three-dimensional structure, are seen as emerging multifunctional cellular components of proteomes. They are highly abundant in eukaryotic proteomes, and numerous functions for these proteins have been proposed. Here, we discuss several reasons why the collection of intrinsically disordered proteins in a proteome (disordome) could be subjected to an active regulation during conditions of water scarcity in plants. We also discuss the potential misinterpretations of disordome content estimations made so far due to bias-prone data and the need for reliable analysis based on experimental data in order to acknowledge the plasticity nature of the disordome.

Evaluation of massive transfusion protocol practices by type of trauma at a level I trauma center.

PURPOSE: To evaluate massive transfusion protocol practices by trauma type at a level I trauma center. METHODS: A retrospective analysis was performed on a sample of 76 trauma patients with MTP activation between March 2010 and January 2015 at a regional trauma center. Patient demographics, transfusion practices, and clinical outcomes were compared by type of trauma sustained. RESULTS: Penetrating trauma patients who required MTP activation were significantly younger, had lower injury severity score (ISS), higher probability of survival (POS), decreased mortality, and higher Glasgow Coma scale (GCS) compared to blunt trauma patients. Overall, the mortality rate was 38.16%. The most common injury sustained among blunt trauma patients was head injury (36.21%), whereas the majority of the penetrating trauma patients sustained abdominal injuries (55.56%). Although the admission coagulation parameters and timing of coagulopathy were not significantly different between the two groups of patients, a significantly higher proportion of penetrating trauma patients received high plasma content therapy relative to blunt trauma patients (p < 0.01). CONCLUSION: Despite the use of the same MTP for all injured patients requiring massive transfusion, significant differences existed between blunt trauma patients and penetrating trauma patients. These differences in transfusion characteristics and outcomes following MTP activation underscore the complexity of implementing MTPs and warrant vigilant transfusion practices to improve outcomes in trauma patients.

Transcriptional profiling of sugarcane leaves and roots under progressive osmotic stress reveals a regulated coordination of gene expression in a spatiotemporal manner.

Sugarcane is one of the most important crops worldwide and is a key plant for the global production of sucrose. Sugarcane cultivation is severely affected by drought stress and it is considered as the major limiting factor for their productivity. In recent years, this plant has been subjected to intensive research focused on improving its resilience against water scarcity; particularly the molecular mechanisms in response to drought stress have become an underlying issue for its improvement. To better understand water stress and the molecular mechanisms we performed a de novo transcriptomic assembly of sugarcane (var. Mex 69-290). A total of 16 libraries were sequenced in a 2x100 bp configuration on a HiSeq-Illumina platform. A total of 536 and 750 genes were differentially up-regulated along with the stress treatments for leave and root tissues respectively, while 1093 and 531 genes were differentially down-regulated in leaves and roots respectively. Gene Ontology functional analysis showed that genes related to response of water deprivation, heat, abscisic acid, and flavonoid biosynthesis were enriched during stress treatment in our study. The reliability of the observed expression patterns was confirmed by RT-qPCR. Additionally, several physiological parameters of sugarcane were significantly affected due to stress imposition. The results of this study may help identify useful target genes and provide tissue-specific data set of genes that are differentially expressed in response to osmotic stress, as well as a complete analysis of the main groups is significantly enriched under this condition. This study provides a useful benchmark for improving drought tolerance in sugarcane and other economically important grass species.

RAP2.4a Is Transported through the Phloem to Regulate Cold and Heat Tolerance in Papaya Tree (Carica papaya cv. Maradol): Implications for Protection Against Abiotic Stress.

Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from Carica papaya cv. Maradol. We isolated four genes (CpRap2.4a, CpRAap2.4b, CpRap2.1 and CpRap2.10), and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2) domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.

Fibrillarin from Archaea to human.

Fibrillarin is an essential protein that is well known as a molecular marker of transcriptionally active RNA polymerase I. Fibrillarin methyltransferase activity is the primary known source of methylation for more than 100 methylated sites involved in the first steps of preribosomal processing and required for structural ribosome stability. High expression levels of fibrillarin have been observed in several types of cancer cells, particularly when p53 levels are reduced, because p53 is a direct negative regulator of fibrillarin transcription. Here, we show fibrillarin domain conservation, structure and interacting molecules in different cellular processes as well as with several viral proteins during virus infection.

Expression of rna polymerase IV and V in oryza sativa

RNA polymerase IV and V are principal players in the RdDM pathway, where their current study has shown interaction of several factors that control DNA silencing of intergenic regions and siRNA production. DNA silencing is an important process during cell differentiation, nuclear structure and viral control. However, RNA pol IV and V are yet to be study in model monocot systems like Oryza sativa that can provide further information on genetic silence mechanism in plats. We show the expression pattern of these polymerases in tissues extracts of Oryza sativa. Detectable amounts of these polymerases are found in specific adult plant tissues and particularly expressed during somatic embryogenesis but not during early stages of normal embryo development. The use of synthetic auxin leads to an induction of both RNA pol IV and V in scutellum tissue where nuclear localization may be required for genome reorganization and gene silencing.

An easy approach for the purification of native TFIIH.

Transcriptional regulation depends on the appropriate set of positive and negative regulating signals in order to provide the correct gene expression. In vitro studies in eukaryotic gene expression over the last few years have provided a wealth of information about new factors involved in the regulation of genes. However, the dissection of this mechanism requires the addition of well-characterized general transcription factors; with the exception of TFIID and TFIIH, all others can easily be expressed in a recombinant form. Here we provide a simple methodology to obtain partially purified transcriptionally active TFIIH free from other general transcription factors and active in transcription.