Panicle transcriptome of high-yield mutant indica rice reveals physiological mechanisms and novel candidate regulatory genes for yield under reproductive stage drought stress.

BACKGROUND: Reproductive stage drought stress (RDS) is a major global threat to rice production. Due to climate change, water scarcity is becoming an increasingly common phenomenon in major rice-growing areas worldwide. Understanding RDS mechanisms will allow candidate gene identification to generate novel rice genotypes tolerant to RDS. RESULTS: To generate novel rice genotypes that can sustain yield under RDS, we performed gamma-irradiation mediated mutation breeding in the drought stress susceptible mega rice variety, MTU1010. One of the mutant MM11 (MTU1010 derived mutant11) shows consistently increased performance in yield-related traits under field conditions consecutively for four generations. In addition, compared to MTU1010, the yield of MM11 is sustained in prolonged drought imposed during the reproductive stage under field and in pot culture conditions. A comparative emerged panicle transcriptome analysis of the MTU1010 and MM11 suggested metabolic adjustment, enhanced photosynthetic ability, and hormone interplay in regulating yield under drought responses during emerged panicle development. Regulatory network analysis revealed few putative significant transcription factor (TF)-target interactions involved in integrated signalling between panicle development, yield and drought stress. CONCLUSIONS: A gamma-irradiate rice mutant MM11 was identified by mutation breeding, and it showed higher potential to sustain yield under reproductive stage drought stress in field and pot culture conditions. Further, a comparative panicle transcriptome revealed significant biological processes and molecular regulators involved in emerged panicle development, yield and drought stress integration. The study extends our understanding of the physiological mechanisms and candidate genes involved in sustaining yield under drought stress.

Structural and Functional Attributes of Microrchidia Family of Chromatin Remodelers.

Chromatin remodelers affect the spatio-temporal dynamics of global gene-expression by structurally modulating and/or reorganizing the chromatin. Microrchidia (MORC) family is a relatively new addition to the four well studied families of chromatin remodeling proteins. In this review, we discuss the current understanding of the structural aspects of human MORCs as well as their epigenetic functions. From a molecular and systems-level perspective, we explore their participation in phase-separated structures, possible influence on various biological processes through protein-protein interactions, and potential extra-nuclear roles. We describe how dysregulation/dysfunction of MORCs can lead to various pathological conditions. We conclude by emphasizing the importance of undertaking integrated efforts to obtain a holistic understanding of the various biological roles of MORCs.

Structure, Activity and Function of the Protein Arginine Methyltransferase 6.

Members of the protein arginine methyltransferase (PRMT) family methylate the arginine residue(s) of several proteins and regulate a broad spectrum of cellular functions. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that asymmetrically dimethylates the arginine residues of numerous substrate proteins. PRMT6 introduces asymmetric dimethylation modification in the histone 3 at arginine 2 (H3R2me2a) and facilitates epigenetic regulation of global gene expression. In addition to histones, PRMT6 methylates a wide range of cellular proteins and regulates their functions. Here, we discuss (i) the biochemical aspects of enzyme kinetics, (ii) the structural features of PRMT6 and (iii) the diverse functional outcomes of PRMT6 mediated arginine methylation. Finally, we highlight how dysregulation of PRMT6 is implicated in various types of cancers and response to viral infections.

PRMT3 interacts with ALDH1A1 and regulates gene-expression by inhibiting retinoic acid signaling.

Protein arginine methyltransferase 3 (PRMT3) regulates protein functions by introducing asymmetric dimethylation marks at the arginine residues in proteins. However, very little is known about the interaction partners of PRMT3 and their functional outcomes. Using yeast-two hybrid screening, we identified Retinal dehydrogenase 1 (ALDH1A1) as a potential interaction partner of PRMT3 and confirmed this interaction using different methods. ALDH1A1 regulates variety of cellular processes by catalyzing the conversion of retinaldehyde to retinoic acid. By molecular docking and site-directed mutagenesis, we identified the specific residues in the catalytic domain of PRMT3 that facilitate interaction with the C-terminal region of ALDH1A1. PRMT3 inhibits the enzymatic activity of ALDH1A1 and negatively regulates the expression of retinoic acid responsive genes in a methyltransferase activity independent manner. Our findings show that in addition to regulating protein functions by introducing methylation modifications, PRMT3 could also regulate global gene expression through protein-protein interactions.

The uncharacterized protein FAM47E interacts with PRMT5 and regulates its functions.

Protein arginine methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues in various proteins affecting diverse cellular processes such as transcriptional regulation, splicing, DNA repair, differentiation, and cell cycle. Elevated levels of PRMT5 are observed in several types of cancers and are associated with poor clinical outcomes, making PRMT5 an important diagnostic marker and/or therapeutic target for cancers. Here, using yeast two-hybrid screening, followed by immunoprecipitation and pull-down assays, we identify a previously uncharacterized protein, FAM47E, as an interaction partner of PRMT5. We report that FAM47E regulates steady-state levels of PRMT5 by affecting its stability through inhibition of its proteasomal degradation. Importantly, FAM47E enhances the chromatin association and histone methylation activity of PRMT5. The PRMT5-FAM47E interaction affects the regulation of PRMT5 target genes expression and colony-forming capacity of the cells. Taken together, we identify FAM47E as a protein regulator of PRMT5, which promotes the functions of this versatile enzyme. These findings imply that disruption of PRMT5-FAM47E interaction by small molecules might be an alternative strategy to attenuate the oncogenic function(s) of PRMT5.

Correction to: N6­Adenosine methylation on mRNA is recognized by YTH2 domain protein of human malaria parasite Plasmodium falciparum.

An amendment to this paper has been published and can be accessed via the original article.

N6-Adenosine methylation on mRNA is recognized by YTH2 domain protein of human malaria parasite Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. The N6-methyl adenosine (m6A) is an important epigenetic modification primarily present on mRNA that controls the levels of transcripts and efficiency of translation in eukaryotes. Recently, the dynamics of m6A on mRNAs at all three developmental stages of P. falciparum in RBCs have been profiled; however, the proteins that regulate the m6A containing mRNAs in the parasites are unknown. RESULTS: Using sequence analysis, we computationally identified that the P. falciparum genome encodes two putative YTH (YT521-B Homology) domain-containing proteins, which could potentially bind to m6A containing mRNA. We developed a modified methylated RNA immunoprecipitation (MeRIP) assay using PfYTH2 and find that it binds selectively to m6A containing transcripts. The PfYTH2 has a conserved aromatic amino acid cage that forms the methyl-binding pocket. Through site-directed mutagenesis experiments and molecular dynamics simulations, we show that F98 residue is important for m6A binding on mRNA. Fluorescence depolarization assay confirmed that PfYTH2 binds to methylated RNA oligos with high affinity. Further, MeRIP sequencing data revealed that PfYTH2 has more permissive sequence specificity on target m6A containing mRNA than other known eukaryotic YTH proteins. Taken together, here we identify and characterize PfYTH2 as the major protein that could regulate m6A containing transcripts in P. falciparum. CONCLUSION: Plasmodium spp. lost the canonical m6A-specific demethylases in their genomes, however, the YTH domain-containing proteins seem to be retained. This study presents a possibility that the YTH proteins are involved in post-transcriptional control in P. falciparum, and might orchestrate the translation of mRNA in various developmental stages of P. falciparum. This is perhaps the first characterization of the methyl-reading function of YTH protein in any parasites.

Epitranscriptomic Code and Its Alterations in Human Disease.

Innovations in epitranscriptomics have resulted in the identification of more than 160 RNA modifications to date. These developments, together with the recent discovery of writers, readers, and erasers of modifications occurring across a wide range of RNAs and tissue types, have led to a surge in integrative approaches for transcriptome-wide mapping of modifications and protein-RNA interaction profiles of epitranscriptome players. RNA modification maps and crosstalk between them have begun to elucidate the role of modifications as signaling switches, entertaining the notion of an epitranscriptomic code as a driver of the post-transcriptional fate of RNA. Emerging single-molecule sequencing technologies and development of antibodies specific to various RNA modifications could enable charting of transcript-specific epitranscriptomic marks across cell types and their alterations in disease.

Diversity in αß and ßα Loop Connections in TIM Barrel Proteins: Implications for Stability and Design of the Fold.

The (ßα)8/TIM barrel is one of the most common folds of known protein structures facilitating diverse catalytic functions. The fold is formed by the repetition of the basic ßαß building block in which the ß-strands are followed by α-helices eight times alternating in sequence and structure. αß and ßα loops connecting α-helices to the ß-strands and the ß-strands to the α-helices contribute to stability and function, respectively, an inherent imposition by the TIM barrel architecture itself. In this study, αß and ßα loops from a data set of 430 non-redundant, high-resolution triosephosphate isomerase (TIM) barrels bearing sequence homology of <30% were analyzed for their amino acid propensities, sequence profiles, and positional preferences of amino acids. While the distribution of short connections is significantly higher in αß loops, there appears to be no such preference in ßα loops. Glycine, proline, lysine, and arginine tend to show greater preference to occur in αß loops, whereas serine, threonine, cysteine, tryptophan, and histidine occur more frequently in ßα loops. In addition, striking dissimilarities in sequence and positional preferences of amino acids, especially, in short, αß and ßα loops are observed. Together, the analysis suggests the role for short loops and charged residues in promoting both non-polar and polar interactions and in ß strand registry. The observed diversity, perhaps, dictates the distinct role of αß and ßα loops in stability and function, respectively. In summary, the overall observations and reasoning, in addition to steering protein engineering efforts on TIM barrel design and stabilization can provide the basis for incorporating consensus loop sequences for designing independently folding ßαß modules.

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

Crowding interactions perturb structure and stability by destabilizing the stable core of the α-subunit of tryptophan synthase.

The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.

Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling.

A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D [(15) N,(1) H] HSQC spectrum with two 2D spectra can result in ∼50 % assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12 kDa) and the 29 kDa (268 residue) α-subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions, even prior to complete resonance assignments.