Publications by authors named "Nimisha Sharma"

24 Publications

  • Page 1 of 1

Deletion of the non-essential Rpb9 subunit of RNA polymerase II results in pleiotropic phenotypes in Schizosaccharomyces pombe.

Biochim Biophys Acta Proteins Proteom 2021 Jul 26;1869(7):140654. Epub 2021 Mar 26.

University School of Biotechnology, G.G.S. Indraprastha University, Sector 16C, Dwarka, New Delhi 110078, India. Electronic address:

Schizosaccharomyces pombe RNA polymerase II comprises twelve different subunits. Its Rpb9 subunit comprises 113 amino acids, and is the only non-essential subunit of S. pombe RNA polymerase II. However, its functions have not been studied in S. pombe. The results presented in this study demonstrate that Rpb9 is involved in regulating growth under optimum and certain stress conditions in S. pombe. To further address the role (s) of various domains of this subunit in regulating these phenotypes, deletion mutant analysis was done. We observed that the region spanning 1-74 amino acids, encompassing the amino-terminal zinc finger domain and the linker region of Rpb9 was able to rescue the phenotypes associated with rpb9deletion. We also demonstrate that the functions of this subunit are only partially conserved among yeast and humans. Our computational biology approaches provide a structural basis for the differential role of various Rpb9 domains in S. pombe. Furthermore, using these tools we show that there has been a co-evolution of the interaction residues between the Rpb9 subunit and the two largest subunits of RNA polymerase II, allowing for a more stringent organism-specific packing. Taken together, our results have provided functional and structural insights into the Rpb9 subunit of S. pombe.
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http://dx.doi.org/10.1016/j.bbapap.2021.140654DOI Listing
July 2021

Functional interaction between ELL transcription elongation factor and Epe1 reveals the role of Epe1 in the regulation of transcription outside heterochromatin.

Mol Microbiol 2021 Feb 3. Epub 2021 Feb 3.

University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India.

Eleven-nineteen lysine-rich leukemia (ELL) is a eukaryotic RNA polymerase II transcription elongation factor. In Schizosaccharomyces pombe, it is important for survival under genotoxic stress conditions. However, the molecular basis underlying this function of ELL in S. pombe is yet to be deciphered. Here, we carried out a genetic screen to identify multicopy suppressor(s) that could restore normal growth of ell1 deletion mutant in the presence of DNA damaging agent. Sequence analysis of the identified suppressors revealed the anti-silencing protein, Epe1, as one of the suppressors of ell1 deletion associated genotoxic stress sensitivity. Our results further demonstrate that the overexpression of Epe1 could suppress all other phenotypes associated with the absence of Ell1. Moreover, transcriptional defect of ell1Δ strain could also be alleviated by the overexpression of Epe1. Epe1 also showed a physical interaction with Ell1. Interestingly, we also observed that the region of Epe1 encompassing 403-948 amino acids was indispensable for all the above functions. Furthermore, our results show that the overexpression of Epe1 causes increased H3K9 acetylation and RNA polymerase II recruitment. Taken together, our results show a functional interaction between Epe1 and Ell1, and this function is independent of the well-known JmjC and N-terminal transcriptional activation domains of Epe1 in S. pombe.
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http://dx.doi.org/10.1111/mmi.14691DOI Listing
February 2021

Comparative RNA sequencing based transcriptome profiling of regular bearing and alternate bearing mango (Mangifera indica L.) varieties reveals novel insights into the regulatory mechanisms underlying alternate bearing.

Biotechnol Lett 2020 Jun 19;42(6):1035-1050. Epub 2020 Mar 19.

ICAR-National Institute for Plant Biotechnology, Pusa campus, New Delhi, 110012, India.

Objective: This study is to understand a comprehensive perspective on the molecular mechanisms underlying alternate bearing in mango (Mangifera indica L.) via transcriptome wide gene expression profiling of both regular and irregular mango varieties.

Results: Transcriptome data of regular (Neelam) and irregular (Dashehari) mango varieties revealed a total of 42,397 genes. Out of that 12,557 significantly differentially expressed genes were identified, of which 6453 were found to be up-regulated and 6104 were found to be down-regulated genes. Further, many of the common unigenes which were involved in hormonal regulation, metabolic processes, oxidative stress, ion homeostasis, alternate bearing etc. showed significant differences between these two different bearing habit varieties. Pathway analysis showed the highest numbers of differentially expressed genes were related with the metabolic processes (523). A total of 26 alternate bearing genes were identified and principally three genes viz; SPL-like gene (GBVX01015803.1), Rumani GA-20-oxidase-like gene (GBVX01019650.1) and LOC103420644 (GBVX01016070.1) were significantly differentially expressed (at log2FC and pval less than 0.05) while, only single gene (gbGBVW01004309.1) related with flowering was found to be differentially expressed. A total of 15 differentially expressed genes from three important pathways viz; alternate bearing, carbohydrate metabolism and hormone synthesis were validated using Real time PCR and results were at par with in silico analysis.

Conclusions: Deciphering the differentially expressed genes (DEGs) and potential candidate genes associated with alternate bearing, hormone and carbohydrate metabolism pathways will help for illustrating the molecular mechanisms underlying the bearing tendencies in mango.
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http://dx.doi.org/10.1007/s10529-020-02863-8DOI Listing
June 2020

A Clinicopathological Correlation of International Federation of Gynecology and Obstetrics's PALM-COEIN Classification of Abnormal Uterine Bleeding: Indian Scenario.

J Midlife Health 2019 Jul-Sep;10(3):147-152

Department of Community Medicine, ESIC Medical College, Faridabad, Haryana, India.

Background: Abnormal uterine bleeding (AUB) is a common problem affecting the women of reproductive age group and may also have a significant impact on their physical, social, and emotional aspects directly affecting their quality of life. The International Federation of Gynecology and Obstetrics (FIGO) devised a universally acceptable system of nomenclature and classification, namely PALM-COEIN classification of AUB in the year 2011. The objective of the present study was to analyze the structural (PALM) and functional (COEIN) component of FIGO system in the Indian scenario.

Materials And Methods: Three hundred patients with complaints of AUB were taken. A clinical diagnosis according to PALM-COEIN system was made after thorough history and clinical examination. Additional investigations if required were done, and endometrial sampling or hysterectomy was done whichever indicated. A histological diagnosis was made, and each case was allocated a category according to PALM-COEIN classification. A clinicopathological correlation was done in the hysterectomy cases for structural causes (PALM).

Results: Leiomyoma (30%) was the most common cause of AUB closely followed by adenomyosis (29.66%) overall. The clinicopathological correlation in hysterectomy cases was good with concordance rate of 85.03%. The concordance between clinical and pathological diagnoses for AUB-L, AUB-A, AUB-M, and AUB-A, L was statistically significant with < 05 in positive cases. However, additional finding of adenomyosis was diagnosed in 48.2% of the cases apart from primary clinical diagnosis.

Conclusion: A good clinicopathological correlation was seen in the cases when classified according to PALM-COEIN classification. The system also provides for consideration of multiple etiologies contributing toward AUB both clinically and histopathologically. However, histopathology remains the cornerstone in establishing the accurate diagnosis as the cases without specific symptoms can be missed clinically.
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http://dx.doi.org/10.4103/jmh.JMH_128_18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767954PMC
October 2019

IAC standardized reporting of breast fine-needle aspiration cytology, Yokohama 2016: A critical appraisal over a 2 year period.

Breast Dis 2019 ;38(3-4):109-115

Department of Pathology, ESIC Medical College & Hospital, NIT 3, Faridabad, India.

Background: Breast cytology is a significant component of the "Triple approach" for pre-operative diagnosis of breast lumps, the other two being clinical assessment and radiological imaging. The role of Fine needle aspiration cytology (FNAC) as a first line investigation in diagnosing breast lesions is well documented, however histopathology is the gold standard. Cyto-histopathological correlation is of great relevance and also increases precision.AIMS \& OBJECTIVES:The present study was conducted with the aim to categorize breast lesions according to the latest standardized reporting system proposed by International academy of cytologists (IAC) in 2016. Evaluation of diagnostic accuracy, sensitivity and specificity of FNAC in diagnosing breast lesions and cyto-histopathological correlation was planned.

Materials And Methods: All FNAs of breast lesions over a period of 2 years were included in the study. The cases were grouped into five standardized categories proposed by the International academy of cytology: Category I (Insufficient material), Category II (Benign), Category III (Atypical, probably benign), Category IV (Suspicious, probably in situ or invasive) & Category V (Malignant) respectively. Specificity, sensitivity, diagnostic accuracy, negative and positive predictive value of FNAC were calculated and cyto-histopathological correlation assessed wherever possible.

Results: Out of 468 breast lesions reported on FNAC, the category wise distribution was - Category I, II, III, IV & V accounting for 23(4.9%), 342(73.07%), 7(1.5%), 11(2.35%) and 85(18.16%) respectively. Histopathology was performed in 331/468 cases with cyto histological concordance of 98.4% and a type agreement rate of 90.9%. The sensitivity, specificity, positive and negative predictive value and diagnostic accuracy was 98.90%, 99.16%, 97.82%, 99.58% and 99.09% respectively.

Conclusion: FNAC is a simple, reliable, cost effective, first line diagnostic procedure for all breast lumps. In collaboration with physical examination and imaging studies (triple approach), FNAC is a highly sensitive diagnostic tool. Adopting a universally acceptable standardized reporting system for breast cytology can enhance the diagnostic accuracy of FNAC.
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http://dx.doi.org/10.3233/BD-190393DOI Listing
March 2020

Basaloid Squamous Cell Carcinoma of Tonsil: an Unusual and Aggressive Variant.

Indian J Surg Oncol 2019 Mar 15;10(1):80-82. Epub 2018 Oct 15.

Department of Pathology, ESIC Medical College and Hospital, Faridabad, Haryana India.

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http://dx.doi.org/10.1007/s13193-018-0816-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414582PMC
March 2019

A comparative study of the proteome regulated by the Rpb4 and Rpb7 subunits of RNA polymerase II in fission yeast.

J Proteomics 2019 05 9;199:77-88. Epub 2019 Mar 9.

University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India. Electronic address:

RNA polymerase II is a conserved multi-subunit enzyme made up of twelve different subunits. Two of these subunits, Rpb4 and Rpb7, have been shown to perform functions in both transcription as well as outside of transcription in Saccharomyces cerevisiae. However, our knowledge about the roles of these subunits in Schizosaccharomyces pombe and higher eukaryotes is still limited. Moreover, both Rpb4 and Rpb7 are indispensable for viability of S. pombe and higher eukaryotes, in comparison to S. cerevisiae where deletion of only Rpb7 results in lethality. Therefore in this study, we used S. pombe strains expressing reduced levels of these subunits to determine their impact on the S. pombe proteome employing i-TRAQ based proteomics approach. Furthermore, proteomic profiling was carried out at two different time points to gain a temporal insight into the processes regulated by Rpb4 and Rpb7. The results showed that reduced levels of either Rpb4 or Rpb7 affected the expression of proteins involved in metabolism and ribosome biogenesis at both the time points. Our polysomal profiling experiments further revealed a role of these subunits in translation. Taken together, our results suggest a key role of Rpb4 and Rpb7 subunits in ribosome biogenesis and protein translation in S. pombe. SIGNIFICANCE: Rpb4 and Rpb7 subunits of RNA polymerase II are known for their diverse roles in regulating transcription, mRNA export, mRNA decay, stress response and translation in S. cerevisiae. However, their roles in other organisms are yet to be characterized in detail. Different lines of evidence also suggest that these subunits may function independently as well as a complex in budding yeast. Therefore, in the present study we employed a genome-wide quantitative proteomics-based approach to gain deeper insights into their cellular roles, and to examine if they regulate similar or different biological pathways in fission yeast. Our results provide evidence that they are both involved in primarily regulating metabolic pathways and ribosome biogenesis and also, play a role in protein translation in S. pombe.
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http://dx.doi.org/10.1016/j.jprot.2019.03.007DOI Listing
May 2019

Are all Granulomatous Mastitis Cases Tuberculous?: A Study on the Role of Cytology in Evaluation of Granulomatous Mastitis.

Turk Patoloji Derg 2019 ;35(2):128-133

Department of Pathology, ESIC Medical College and Hospital, NIT 3, FARIDABAD, INDIA.

Objective: Granulomatous mastitis is a rare inflammatory condition of the breast clinically mimicking breast carcinoma and pyogenic abscess, thereby creating a diagnostic dilemma. Tuberculous mastitis is a rare clinical entity accounting for approximately 3% of all mammary lesions. All cases of granulomatous mastitis diagnosed cytologically over a period of 3 years were evaluated to ascertain the cases with tuberculous etiology.

Material And Method: Fine needle aspiration cytology of the breast lump was performed and all the cytological parameters were evaluated. Wherever histopathology was available, the diagnosis was confirmed on Hematoxylin & Eosin stained sections and the Ziehl Neelsen (ZN) stain was applied.

Results: A total of 10 cases of granulomatous mastitis were diagnosed on cytology during the 3-year period. On cytological smears, four cases showed presence of ill-formed granulomas and one case had scattered epithelioid histiocytes; however, the rest of the cases had well-formed granulomas. Finally, four out of ten cases were labeled as tuberculous mastitis based on the ZN stain/Tuberculosis-Polymerase chain reaction (TB-PCR) and the other six cases were granulomatous mastitis.

Conclusion: Cytology plays a significant role in the diagnosis of granulomatous mastitis. This study re-emphasizes the role of the cytopathologist in the accurate and early diagnosis of these lesions so that unnecessary surgery can be avoided, and also highlights the fact that all granulomatous mastitis cases are not tuberculous.
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http://dx.doi.org/10.5146/tjpath.2018.01442DOI Listing
September 2019

Genome-wide transcriptional response to altered levels of the Rpb7 subunit of RNA polymerase II identifies its role in DNA damage response in Schizosaccharomyces pombe.

FEMS Yeast Res 2019 01;19(1)

University School of Biotechnology, G.G.S. Indraprastha University, Sector 16C, Dwarka, New Delhi 110078, India.

Transcription of protein-coding genes is a highly regulated process. In eukaryotes, it involves cross-talk between the RNA polymerase II enzyme and different proteins of the transcriptional machinery. Twelve different subunits, Rpb1 to Rpb12, constitute RNA polymerase II. The sequence of the Rpb7 subunit is highly conserved across organisms. However, our knowledge and understanding of the role of Rpb7 in Schizosaccharomyces pombe is still limited. Therefore, in the present study we have studied the transcriptional response of S. pombe cells expressing reduced levels of rpb7+. Our global transcriptional analysis revealed that expression of genes belonging to different DNA repair pathways was downregulated by reduced rpb7+ expression. It was observed that survival of S. pombe cells expressing low rpb7+ levels was compromised under genotoxic stress conditions. Rpb7 also exhibited genetic interaction with genes of various DNA repair pathways. Furthermore, the growth sensitivity of S. pombe cells with low rpb7+ levels under DNA-damaging conditions was completely rescued by human Rpb7, indicating a functional conservation between these proteins. In summary, results from our whole-genome level gene expression analysis, as well as phenotypic and genetic experiments suggest a role for Rpb7 in DNA damage response in S. pombe.
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http://dx.doi.org/10.1093/femsyr/foy118DOI Listing
January 2019

Schizosaccharomyces pombe Pol II transcription elongation factor ELL functions as part of a rudimentary super elongation complex.

Nucleic Acids Res 2018 11;46(19):10095-10105

Stowers Institute for Medical Research, Kansas City, MO 64110, USA.

ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.
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http://dx.doi.org/10.1093/nar/gky713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212713PMC
November 2018

MiSNPDb: a web-based genomic resources of tropical ecology fruit mango (Mangifera indica L.) for phylogeography and varietal differentiation.

Sci Rep 2017 11 2;7(1):14968. Epub 2017 Nov 2.

ICAR-National Research Centre on Plant Biotechnology, New Delhi, India.

Mango is one of the most important fruits of tropical ecological region of the world, well known for its nutritive value, aroma and taste. Its world production is >45MT worth >200 billion US dollars. Genomic resources are required for improvement in productivity and management of mango germplasm. There is no web-based genomic resources available for mango. Hence rapid and cost-effective high throughput putative marker discovery is required to develop such resources. RAD-based marker discovery can cater this urgent need till whole genome sequence of mango becomes available. Using a panel of 84 mango varieties, a total of 28.6 Gb data was generated by ddRAD-Seq approach on Illumina HiSeq 2000 platform. A total of 1.25 million SNPs were discovered. Phylogenetic tree using 749 common SNPs across these varieties revealed three major lineages which was compared with geographical locations. A web genomic resources MiSNPDb, available at http://webtom.cabgrid.res.in/mangosnps/ is based on 3-tier architecture, developed using PHP, MySQL and Javascript. This web genomic resources can be of immense use in the development of high density linkage map, QTL discovery, varietal differentiation, traceability, genome finishing and SNP chip development for future GWAS in genomic selection program. We report here world's first web-based genomic resources for genetic improvement and germplasm management of mango.
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http://dx.doi.org/10.1038/s41598-017-14998-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668432PMC
November 2017

The amino-terminal domain of ELL transcription elongation factor is essential for ELL function in Schizosaccharomyces pombe.

Microbiology (Reading) 2017 Nov 18;163(11):1641-1653. Epub 2017 Oct 18.

University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi-110078, India.

Transcriptional elongation is a critical step for regulating expression of protein-coding genes. Multiple transcription elongation factors have been identified in vitro, but the physiological roles of many of them are still not clearly understood. The ELL (Eleven nineteen Lysine rich Leukemia) family of transcription elongation factors are conserved from fission yeast to humans. Schizosaccharomyces pombe contains a single ELL homolog (SpELL) that is not essential for its survival. Therefore to gain insights into the in vivo cellular functions of SpELL, we identified phenotypes associated with deletion of ell1 in S. pombe. Our results demonstrate that SpELL is required for normal growth of S. pombe cells. Furthermore, cells lacking ell1 exhibit a decrease in survival when exposed to DNA-damaging conditions, but their growth is not affected under environmental stress conditions. ELL orthologs in different organisms contain three conserved domains, an amino-terminal domain, a middle domain and a carboxyl-terminal domain. We also carried out an in vivo functional mapping of these conserved domains within S. pombe ELL and uncovered a critical role for its amino-terminus in regulating all its cellular functions, including growth under different conditions, transcriptional elongation potential and interaction with S. pombe EAF. Taken together our results suggest that the domain organization of ELL proteins is conserved across species, but the in vivo functions as well as the relationship between the various domains and roles of ELL show species-specific differences.
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http://dx.doi.org/10.1099/mic.0.000554DOI Listing
November 2017

Structure function characterization of the ELL Associated Factor (EAF) from Schizosaccharomyces pombe.

Gene 2018 Jan 13;641:117-128. Epub 2017 Oct 13.

University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India. Electronic address:

EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.
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http://dx.doi.org/10.1016/j.gene.2017.10.031DOI Listing
January 2018

Apocrine carcinoma of breast: A rare entity posing cytological challenge.

Diagn Cytopathol 2017 Dec 21;45(12):1156-1158. Epub 2017 Sep 21.

Department of Surgery, ESIC Medical College, Faridabad, Haryana, India.

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http://dx.doi.org/10.1002/dc.23819DOI Listing
December 2017

Leaf Transcriptome Sequencing for Identifying Genic-SSR Markers and SNP Heterozygosity in Crossbred Mango Variety 'Amrapali' (Mangifera indica L.).

PLoS One 2016 13;11(10):e0164325. Epub 2016 Oct 13.

ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, India.

Mango (Mangifera indica L.) is called "king of fruits" due to its sweetness, richness of taste, diversity, large production volume and a variety of end usage. Despite its huge economic importance genomic resources in mango are scarce and genetics of useful horticultural traits are poorly understood. Here we generated deep coverage leaf RNA sequence data for mango parental varieties 'Neelam', 'Dashehari' and their hybrid 'Amrapali' using next generation sequencing technologies. De-novo sequence assembly generated 27,528, 20,771 and 35,182 transcripts for the three genotypes, respectively. The transcripts were further assembled into a non-redundant set of 70,057 unigenes that were used for SSR and SNP identification and annotation. Total 5,465 SSR loci were identified in 4,912 unigenes with 288 type I SSR (n ≥ 20 bp). One hundred type I SSR markers were randomly selected of which 43 yielded PCR amplicons of expected size in the first round of validation and were designated as validated genic-SSR markers. Further, 22,306 SNPs were identified by aligning high quality sequence reads of the three mango varieties to the reference unigene set, revealing significantly enhanced SNP heterozygosity in the hybrid Amrapali. The present study on leaf RNA sequencing of mango varieties and their hybrid provides useful genomic resource for genetic improvement of mango.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164325PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063295PMC
May 2017

Regulation of RNA polymerase II-mediated transcriptional elongation: Implications in human disease.

Authors:
Nimisha Sharma

IUBMB Life 2016 09 29;68(9):709-16. Epub 2016 Jul 29.

University School of Biotechnology, G.G.S. Indraprastha University, Dwarka, New Delhi, 110078, India.

Expression of protein-coding genes is primarily regulated at the level of transcription. Most of the earlier studies focussed on understanding the assembly of the pre-initiation complex at the promoter of genes and subsequent initiation of transcription as the regulatory steps in transcription. However, research over the last decade has demonstrated the significance of regulating transcription of genes at the elongation stage. Several new proteins have been identified that control this step and our knowledge about their functions is expanding rapidly. Moreover, an increasing body of evidence suggests that a dysfunction of these transcription elongation factors is related to several diseases. Here, we review the latest advances in our understanding about the in vivo roles of the transcription elongation factors and their link with diseases. © 2016 IUBMB Life, 68(9):709-716, 2016.
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http://dx.doi.org/10.1002/iub.1538DOI Listing
September 2016

Mechanism of Ca²⁺-triggered ESCRT assembly and regulation of cell membrane repair.

Nat Commun 2014 Dec 23;5:5646. Epub 2014 Dec 23.

1] Children's National Medical Center, Center for Genetic Medicine Research, 111 Michigan Avenue, NW, Washington DC 20010-2970, USA [2] Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington DC, USA.

In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.
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http://dx.doi.org/10.1038/ncomms6646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333728PMC
December 2014

Modulating the level of the Rpb7 subunit of RNA polymerase II affects cell separation in Schizosaccharomyces pombe.

Res Microbiol 2015 Jan 18;166(1):20-7. Epub 2014 Dec 18.

University School of Biotechnology, G.G.S.Indraprastha University, Sector 16C, Dwarka, New Delhi 110078, India. Electronic address:

The rpb7(+) gene encodes the seventh largest subunit of RNA polymerase II and is essential for survival of yeast cells. To gain insight into its functions, we expressed rpb7(+) under the control of the nmt1 promoter and investigated its role in regulating multiple phenotypes in Schizosaccharomyces pombe. We observed that low rpb7(+) levels resulted in slow growth of cells under optimum growth conditions. However, no growth defect was observed under different stress conditions tested in this study. Our results also showed that the most prominent phenotype of cells expressing reduced rpb7(+) is a defect in cell separation. Quantitative real-time PCR analysis further revealed that the transcription of specific cell septation genes was significantly reduced in these cells. Collectively, results presented in this study highlight the distinct role of Rpb7p in regulating cell separation in S. pombe.
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http://dx.doi.org/10.1016/j.resmic.2014.12.002DOI Listing
January 2015

Rpb4 and Rpb7: multifunctional subunits of RNA polymerase II.

Crit Rev Microbiol 2013 Nov 24;39(4):362-72. Epub 2012 Aug 24.

University School of Biotechnology, G. G. S. Indraprastha University , Dwarka, New Delhi , India.

The 12-subunit RNA polymerase II enzyme in yeasts and higher eukaryotic cells is important for transcription of protein-coding genes. Its fourth and seventh largest subunits named Rpb4 and Rpb7, respectively, display some unique features that distinguish them from the remaining subunits of this enzyme. These two subunits also bind to each other forming a complex in archaebacteria, yeasts, plants and humans. Our knowledge about the structure and functions of this complex has greatly advanced in recent years. These subunits were initially considered to be important only for initiation of transcription and stress response. However, recent evidence suggests that they are not only involved in transcription, but also in DNA repair, mRNA export and decay as well as translation, highlighting the roles of this heterodimer in diverse biological processes. In this article, we review the current status of these two subunits and discuss attributes of their structure and function across organisms.
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http://dx.doi.org/10.3109/1040841X.2012.711742DOI Listing
November 2013

Use of quantitative membrane proteomics identifies a novel role of mitochondria in healing injured muscles.

J Biol Chem 2012 Aug 9;287(36):30455-67. Epub 2012 Jul 9.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC 20010, USA.

Skeletal muscles are proficient at healing from a variety of injuries. Healing occurs in two phases, early and late phase. Early phase involves healing the injured sarcolemma and restricting the spread of damage to the injured myofiber. Late phase of healing occurs a few days postinjury and involves interaction of injured myofibers with regenerative and inflammatory cells. Of the two phases, cellular and molecular processes involved in the early phase of healing are poorly understood. We have implemented an improved sarcolemmal proteomics approach together with in vivo labeling of proteins with modified amino acids in mice to study acute changes in the sarcolemmal proteome in early phase of myofiber injury. We find that a notable early phase response to muscle injury is an increased association of mitochondria with the injured sarcolemma. Real-time imaging of live myofibers during injury demonstrated that the increased association of mitochondria with the injured sarcolemma involves translocation of mitochondria to the site of injury, a response that is lacking in cultured myoblasts. Inhibiting mitochondrial function at the time of injury inhibited healing of the injured myofibers. This identifies a novel role of mitochondria in the early phase of healing injured myofibers.
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http://dx.doi.org/10.1074/jbc.M112.354415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436295PMC
August 2012

The Med8 mediator subunit interacts with the Rpb4 subunit of RNA polymerase II and Ace2 transcriptional activator in Schizosaccharomyces pombe.

FEBS Lett 2009 Oct 29;583(19):3115-20. Epub 2009 Aug 29.

University School of Biotechnology, G.G.S. Indraprastha University, Kashmere Gate, Delhi 110 403, India.

Several proteins are involved in separation of cells following division. However, their mutual interactions leading to cell separation is complex and not well understood. To explore the protein network that regulates this process at the transcriptional level in Schizosaccharomyces pombe, we have investigated the role of three proteins Med8, Rpb4 and Ace2. Using genetic and biochemical approaches we demonstrate that Ace2 binds Med8, which in turn interacts with Rpb4. We have delineated regions of Med8 and Rpb4 involved in their binding. We show that Med8 carboxyl-terminal region is necessary for its interaction with Rpb4 and can partially complement the sep15-598 mutant. Our results suggest that Med8 mediator subunit is involved in transmitting regulatory information from Ace2 to RNA polymerase II via Rpb4.
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http://dx.doi.org/10.1016/j.febslet.2009.08.036DOI Listing
October 2009

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation.

Mol Genet Genomics 2006 Dec 14;276(6):545-54. Epub 2006 Sep 14.

University School of Biotechnology, G.G.S. Indraprastha University, Kashmere Gate, Delhi, 110006, India.

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.
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http://dx.doi.org/10.1007/s00438-006-0161-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1705487PMC
December 2006

Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae.

Biochem Biophys Res Commun 2005 Jul;332(3):763-70

School of Biotechnology, G.G.S. Indraprastha University, Kashmere Gate, Delhi 110006, India.

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.
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http://dx.doi.org/10.1016/j.bbrc.2005.05.015DOI Listing
July 2005

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani.

EMBO J 2003 Nov;22(21):5712-22

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin-horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgammaS, GTPgammaS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody specific for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.
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http://dx.doi.org/10.1093/emboj/cdg557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC275414PMC
November 2003