DEAD Box RNA Helicases: Biochemical Properties, Role in RNA Processing and Ribosome Biogenesis.

DEAD box RNA helicases are a versatile group of ATP dependent enzymes that play an essential role in cellular processes like transcription, RNA processing, ribosome biogenesis and translation. These enzymes perform structural rearrangement of complex RNA molecules and enhance the productive folding of RNA and organization of macromolecular complexes. In this review article besides providing the outline about structural organization of helicases, an in-depth discussion will be done on the biochemical properties of RNA helicases like their substrate binding, binding and hydrolysis of ATP and related conformational changes that are important for functioning of the RNA helicase enzymes. I will extensively discuss the physiological role of RNA helicases in RNA processing and ribosome biogenesis.

Role of DEAD-box RNA helicases in low-temperature adapted growth of Antarctic Pseudomonas syringae Lz4W.

IMPORTANCE: RNA metabolism is important as RNA acts as a link between genomic information and functional biomolecules, thereby playing a critical role in cellular response to environment. We investigated the role of DEAD-box RNA helicases in low-temperature adapted growth of P. syringae, as this group of enzymes play an essential role in modulation of RNA secondary structures. This is the first report on the assessment of all major DEAD-box RNA helicases in any Antarctic bacterium. Of the five RNA helicases, three (srmB, csdA, and dbpA) are important for the growth of the Antarctic P. syringae at low temperature. However, the requisite role of dbpA and the indispensable requirement of csdA for low-temperature adapted growth are a novel finding of this study. Growth analysis of combinatorial deletion strains was performed to understand the functional interaction among helicase genes. Similarly, genetic complementation of RNA helicase mutants was conducted for identification of gene redundancy in P. syringae.

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress.

IMPORTANCE: Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.

Functional activity of E. coli RNase R in the Antarctic Pseudomonas syringae Lz4W.

BACKGROUND: In Antarctic P. syringae RNase R play an essential role in the processing of 16S and 5S rRNA, thereby playing an important role in cold-adapted growth of the bacterium. This study is focused on deciphering the in vivo functional activity of mesophilic exoribonuclease R and its catalytic domain (RNB) in an evolutionary distant psychrophilic bacterium Pseudomonas syringae Lz4W. RESULTS: Our results confirm that E. coli RNase R complemented the physiological functions of the psychrophilic bacterium P. syringae RNase R and rescued the cold-sensitive phenotype of Pseudomonas syringae ∆rnr mutant. More importantly, the catalytic domain (RNB) of the E. coli RNase R is also capable of alleviating the cold-sensitive growth defects of ∆rnr mutant as seen with the catalytic domain (RNB) of the P. syringae enzyme. The Catalytic domain of E. coli RNase R was less efficient than the Catalytic domain of P. syringae RNase R in rescuing the cold-sensitive growth of ∆rnr mutant at 4°C, as the ∆rnr expressing the RNBEc (catalytic domain of E. coli RNase R) displayed longer lag phase than the RNBPs (Catalytic domain of P. syringae RNase R) complemented ∆rnr mutant at 4°C. Altogether it appears that the E. coli RNase R and P. syringae RNase R are functionally exchangeable for the growth requirements of P. syringae at low temperature (4°C). Our results also confirm that in P. syringae the requirement of RNase R for supporting the growth at 4°C is independent of the degradosomal complex. CONCLUSION: E. coli RNase R (RNase REc) rescues the cold-sensitive phenotype of the P. syringae Δrnr mutant. Similarly, the catalytic domain of E. coli RNase R (RNBEc) is also capable of supporting the growth of Δrnr mutant at low temperatures. These findings have a vast scope in the design and development of low-temperature-based expression systems.

DEAD box RNA helicases protect Antarctic Pseudomonas syringae Lz4W against oxidative stress.

DEAD box RNA helicases are involved in important cellular processes like RNA metabolism (Processing and Degradation), ribosome biogenesis and translation. Besides being crucial to the formation of cold adapted degradosomes, RNA helicases have been implicated in structural rearrangement of RNA, implying a role in alleviation of RNA secondary structure stabilization at low temperature. This study depicts the results of experiments on protective role played by DEAD box RNA helicases against nucleic acid damaging agents. RNA helicase mutants ΔrhlE, ΔsrmB, ΔcsdA, ΔdbpA and ΔrhlB cells were exposed to various DNA damaging agents (UV, Paraquat, Mitomycin C, Hydroxyurea and Hydrogen peroxide) and assessed for sensitivity to them. Our results illustrate that ∆csdA displayed sensitivity to paraquat (that causes oxidative damage) and UV radiation induced DNA damage. On the other hand, ∆srmB displays sensitivity to hydroxyurea that causes damage to the replication forks (RFs) by inhibiting ribonucleotide reductase and depleting the dNTP pool of cells. However, all five RNA helicase mutants were resistant to H2O2 mediated oxidative stress and mitomycin C induced DNA cross-links.

Phylogeny and Optimization of Trichoderma harzianum for Chitinase Production: Evaluation of Their Antifungal Behaviour against the Prominent Soil Borne Phyto-Pathogens of Temperate India.

Trichoderma is the most commonly used fungal biocontrol agent throughout the world. In the present study, various Trichoderma isolates were isolated from different vegetable fields. In the isolated microflora, the colony edges varied from wavy to smooth. The mycelial forms were predominantly floccose with hyaline color and conidiophores among all the strains were highly branched. Based on morphological attributes, all the isolates were identified as Trichoderma harzianum. The molecular identification using multilocus sequencing ITS, rpb2 and tef1α, genes further confirmed the morphological identification. The average chitinase activity varied from 1.13 units/mL to 3.38 units/mL among the various isolates, which increased linearly with temperature from 15 to 30 °C. There was an amplified production in the chitinase production in the presence of Mg+ and Ca2+ and Na+ metal ions, but the presence of certain ions was found to cause the down-regulated chitinase activity, i.e., Zn2+, Hg2+, Fe2+, Ag+ and K+. All the chitinase producing Trichoderma isolates inhibited the growth of tested pathogens viz., Dematophora necatrix, Fusarium solani, Fusarium oxysporum and Pythium aphanidermatum at 25% culture-free filtrate concentration under in vitro conditions. Also, under in vivo conditions, the lowest wilt incidence and highest disease control on Fusarium oxysporum was observed in isolate BT4 with mean wilt incidence and disease control of 21% and 48%, respectively. The Trichoderma harzianum identified in this study will be further used in formulation development for the management of diseases under field conditions.

Molecular phylogeny, pathogenic variability and phytohormone production of Fusarium species associated with bakanae disease of rice in temperate agro-ecosystems.

Bakanae is the emerging disease threating the rice cultivation globally. Yield reduction of 4-70% is recorded in different parts of the world. A total of 119 Fusarium isolates were collected from rice plants at different geographical locations and seeds of different rice cultivars. The isolates were evaluated for morphological, biochemical and pathogenic diversity. The amplification of TEF-1α gene was carried out for exploring the species spectrum associated with the cultivated and pre-released rice varieties. The production of gibberellin varied from 0.53 to 2.26 µg/25 ml, while as that of Indole acetic acid varied from 0.60 to 3.15 µg/25 ml among the Fusarium isolates. The phylogenetic analysis identified 5 different species of the genus Fusarium viz. Fusarium fujikuroi, F. proliferatum, F. equiseti, F.oxysporum and F. persicinum after nucleotide blasting in NCBI. Only two Fusarium spp. F. fujikuroi and F. proliferatum were found to be pathogenic under virulence assays of the isolates. The isolates showed a considerable variation in morphological and pathogenic characters. The isolates were divided into different groups based on morphology and pathogenicity tests. The isolates showed a considerable variation in morphology, phytohormone profile and virulence indicative of population diversity. Three species F. equiseti, F.oxysporum and F. persicinum which have not been reported as pathogens of rice in India were found to be associated with bakanae disease of rice, however their pathogenicity could not be established.

Generation of three spinocerebellar ataxia type-12 patients derived induced pluripotent stem cell lines (IGIBi002-A, IGIBi003-A and IGIBi004-A).

Spinocerebellar ataxia type-12 (SCA12) is a neurological disorder caused due to triplet (CAG) repeat expansion in 5' UTR of PPP2R2B. It is one of the most prominent SCA-subtype in Indian population and till date no patient specific models have been described. Human-induced-pluripotent-stem cell (HiPSC) based disease modelling has become the next generation tool for studying various human pathologies. In the present study we established three SCA12 patient specific iPSC lines. All the generated lines have shown pluripotency markers, normal karyotype, in-vitro three germ layers differentiation potential, vector clearance, SCA12 mutation, parental genomic identity and contamination free culture.