Global Run-On sequencing to measure nascent transcription in Saccharomyces cerevisiae.

Global Run-On sequencing is a reliable and widely used approach for monitoring nascent transcription on a genomewide scale. The assay has been successfully used for studying global transcription in humans, plants, worms, flies, and fission yeast. Here we describe a GRO-seq protocol for studying transcription in budding yeast, Saccharomyces cerevisiae. Briefly, the technique involves permeabilization of actively growing yeast cells, allowing transcription to proceed in permeabilized cells in the presence of brominated UTP, affinity purification of bromo-UMP incorporated nascent transcripts followed by cDNA library construction, deep sequencing, and mapping against the reference genome. The approach maps the position of transcriptionally active RNA polymerase on a genomewide basis. In addition to identifying the complete set of transcriptionally active genes in a cell under a given set of conditions, the method can be used to determine elongation rate, termination defect and promoter directionality at the genomewide level. The approach is especially useful in identifying short-lived unstable transcripts that are rapidly degraded even before they leave the nucleus.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch.

TFIIB-Termination Factor Interaction Affects Termination of Transcription on Genome-Wide Scale.

Apart from its well-established role in the initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remain largely unexplored. Using GRO-seq analyses, we compared the terminator readthrough phenotype in the sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains. Approximately 74% of genes analyzed exhibited a 2-3-fold increase in readthrough of the poly(A)-termination signal in the TFIIBsua7-1 mutant compared to TFIIBWT cells. To understand the mechanistic basis of TFIIB's role in termination, we performed the mass spectrometry of TFIIB-affinity purified from chromatin and soluble cellular fractions-from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with CF1A and Rat1 complexes in the TFIIBsua7-1 mutant. ChIP assays revealed about a 90% decline in the recruitment of termination factors in the TFIIBsua7-1 mutant compared to wild type cells. The overall conclusion of these results is that TFIIB affects the termination of transcription on a genome-wide scale, and the TFIIB-termination factor interaction plays a crucial role in the process.

A termination-independent role of Rat1 in cotranscriptional splicing.

Rat1 is a 5'→3' exoribonuclease in budding yeast. It is a highly conserved protein with homologs being present in fission yeast, flies, worms, mice and humans. Rat1 and its human homolog Xrn2 have been implicated in multiple nuclear processes. Here we report a novel role of Rat1 in mRNA splicing. We observed an increase in the level of unspliced transcripts in mutants of Rat1. Accumulation of unspliced transcripts was not due to the surveillance role of Rat1 in degrading unspliced mRNA, or an indirect effect of Rat1 function in termination of transcription or on the level of splicing factors in the cell, or due to an increased elongation rate in Rat1 mutants. ChIP-Seq analysis revealed Rat1 crosslinking to the introns of a subset of yeast genes. Mass spectrometry and coimmunoprecipitation revealed an interaction of Rat1 with the Clf1, Isy1, Yju2, Prp43 and Sub2 splicing factors. Furthermore, recruitment of splicing factors on the intron was compromised in the Rat1 mutant. Based on these findings we propose that Rat1 has a novel role in splicing of mRNA in budding yeast. Rat1, however, is not a general splicing factor as it crosslinks to only long introns with an average length of 400 nucleotides.

Beyond the canonical role of TFIIB in eukaryotic transcription.

The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5' and 3' ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3' end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3' end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit.

Gene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB-Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB-Ssu72 mediated gene looping.

Enhancement of Transcription by a Splicing-Competent Intron Is Dependent on Promoter Directionality.

Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on (TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts (uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5' or 3' splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5' splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly(A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality.

A role for CF1A 3' end processing complex in promoter-associated transcription.

The Cleavage Factor 1A (CF1A) complex, which is required for the termination of transcription in budding yeast, occupies the 3' end of transcriptionally active genes. We recently demonstrated that CF1A subunits also crosslink to the 5' end of genes during transcription. The presence of CF1A complex at the promoter suggested its possible involvement in the initiation/reinitiation of transcription. To check this possibility, we performed transcription run-on assay, RNAP II-density ChIP and strand-specific RT-PCR analysis in a mutant of CF1A subunit Clp1. As expected, RNAP II read through the termination signal in the temperature-sensitive mutant of clp1 at elevated temperature. The transcription readthrough phenotype was accompanied by a decrease in the density of RNAP II in the vicinity of the promoter region. With the exception of TFIIB and TFIIF, the recruitment of the general transcription factors onto the promoter, however, remained unaffected in the clp1 mutant. These results suggest that the CF1A complex affects the recruitment of RNAP II onto the promoter for reinitiation of transcription. Simultaneously, an increase in synthesis of promoter-initiated divergent antisense transcript was observed in the clp1 mutant, thereby implicating CF1A complex in providing directionality to the promoter-bound polymerase. Chromosome Conformation Capture (3C) analysis revealed a physical interaction of the promoter and terminator regions of a gene in the presence of a functional CF1A complex. Gene looping was completely abolished in the clp1 mutant. On the basis of these results, we propose that the CF1A-dependent recruitment of RNAP II onto the promoter for reinitiation and the regulation of directionality of promoter-associated transcription are accomplished through gene looping.

Role for gene looping in intron-mediated enhancement of transcription.

Intron-containing genes are often transcribed more efficiently than nonintronic genes. The effect of introns on transcription of genes is an evolutionarily conserved feature, being exhibited by such diverse organisms as yeast, plants, flies, and mammals. The mechanism of intron-mediated transcriptional activation, however, is not entirely clear. To address this issue, we inserted an intron in INO1, which is a nonintronic gene, and deleted the intron from ASC1, which contains a natural intron. We then compared transcription of INO1 and ASC1 genes in the presence and absence of an intron. Transcription of both genes was significantly stimulated by the intron. The introns have a direct role in enhancing transcription of INO1 and ASC1 because there was a marked increase in nascent transcripts from these genes in the presence of an intron. Intron-mediated enhancement of transcription required a splicing competent intron. Interestingly, both INO1 and ASC1 were in a looped configuration when their genes contained an intron. Intron-dependent gene looping involved a physical interaction of the promoter and the terminator regions. In addition, the promoter region interacted with the 5' splice site and the terminator with the 3' splice site. Intron-mediated enhancement of transcription was completely abolished in the looping defective sua7-1 strain. No effect on splicing, however, was observed in sua7-1 strain. On the basis of these results, we propose a role for gene looping in intron-mediated transcriptional activation of genes in yeast.

Srb5/Med18-mediated termination of transcription is dependent on gene looping.

We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the termination of transcription for a subset of genes in yeast. Srb5/Med18 could affect termination either indirectly by modulating CTD-Ser(2) phosphorylation near the 3' end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser(2) phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3' end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5(-) cells. Furthermore, Srb5-dependent termination of transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated termination of transcription, and the looped gene architecture may have a general role in termination of transcription in budding yeast.

Ssu72: a versatile protein with functions in transcription and beyond.

Eukaryotic transcription is a complex process involving a vast network of protein and RNA factors that influence gene expression. The main player in transcription is the RNA polymerase that synthesizes the RNA from the DNA template. RNA polymerase II (RNAPII) transcribes all protein coding genes and some noncoding RNAs in eukaryotic cells. The polymerase is aided by interacting partners that shuttle it along the gene for initiation, elongation and termination of transcription. One of the many factors that assist RNAPII in transcription of genes is Ssu72. It is a carboxy-terminal-domain (CTD)-phosphatase that plays pleiotropic roles in the transcription cycle. It is essential for cell viability in Saccharomyces cerevisiae, the organism in which it was discovered. The homologues of Ssu72 have been identified in humans, mice, plants, flies, and fungi thereby suggesting the evolutionarily conserved nature of the protein. Recent studies have implicated the factor beyond the confines of transcription in homeostasis and diseases.

Protein interaction network revealed by quantitative proteomic analysis links TFIIB to multiple aspects of the transcription cycle.

Although TFIIB is widely regarded as an initiation factor, recent reports have implicated it in multiple aspects of eukaryotic transcription. To investigate the broader role of TFIIB in transcription, we performed quantitative proteomic analysis of yeast TFIIB. We purified two different populations of TFIIB; one from soluble cell lysate, which is not engaged in transcription, and the other from the chromatin fraction which yields the transcriptionally active form of the protein. TFIIB purified from the chromatin exhibits several interactions that explain its non-canonical roles in transcription. RNAPII, TFIIF and TFIIH were the only components of the preinitiation complex with a significant presence in chromatin TFIIB. A notable feature was enrichment of all subunits of CF1 and Rat1 3' end processing-termination complexes in chromatin-TFIIB preparation. Subunits of the CPF termination complex were also detected in both chromatin and soluble derived TFIIB preparations. These results may explain the presence of TFIIB at the 3' end of genes during transcription as well as its role in promoter-termination interaction.

Genome-wide analysis of TFIIB's role in termination of transcription.

This study provides evidence that the role of TFIIB extends beyond initiation to include the termination step of transcription. Using GRO-seq analyses, we compared terminator readthrough phenotype in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains. Approximately 74% of genes analyzed exhibited a 2-3-fold increase in readthrough of the poly(A)-termination signal in the TFIIB sua7-1 mutant compared to TFIIB WT cells. Mass spectrometry of affinity purified TFIIB from chromatin fraction found TFIIB exhibiting interaction with CF1A and Rat1 termination complexes in TFIIB WT cells. There was, however, a drastic decrease in TFIIB interaction with CF1A and Rat1 termination complexes in the TFIIB sua7-1 mutant. ChIP assays revealed about 90% decline in recruitment of termination factors in TFIIB sua7-1 mutant compared to wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

Genome-wide analysis of TFIIB's role in termination of transcription.

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB sua7-1 mutant compared to the TFIIB WT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB sua7-1 and TFIIB WT cells. TFIIB purified from the chromatin fraction of TFIIB WT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB sua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

Antipyretic activity of the hydro-alcoholic extract of Artemisia absinthium L. as a standalone and as an adjuvant with barley water against yeast-induced pyrexia in albino Wistar rats.

OBJECTIVES: In Unani medicine, a comprehensive treatment plan has been delineated to deal with febrile illnesses using herbal drugs along with modified dietetics, which stands as a promising area of research. The present study was aimed at evaluating the antipyretic activity of the HAE of Artemisia absinthium L. whole plant as a standalone and as an adjuvant with barley water in an animal model of pyrexia to validate the age-old Unani principle of the treatment. METHODS: The pyrexia was induced in all the groups except the plain control using Brewer's yeast. Group II did not receive any treatment, while group III received crocin, group IV received HAE of A. absinthium, group V administered Ma al-Sha'ir, and group VI was treated with the HAE of A. absinthium along with Ma al-Sha'ir by oral route. The rectal temperature of each rat was recorded at '0' h, 30 min, 60 min, and 180 min. RESULTS: The mean rectal temperature of group III went down from 101.82±0.20 °F to 100.4±0.57 °F over the period of (0-180) minutes, whereas the mean temperature in group IV went down from 102.45±0.60 °F to 100.14±0.57 °F. The mean rectal temperature of group V decreased from 100.62±0.11 °F to 99.55±0.51 °F, while the mean rectal temperature of group VI went down from 101.95±0.1 °F to 97.7±0.11 °F. CONCLUSIONS: It is concluded that the HAE of A. absinthium L. as a standalone and along with Ma al Sha'ir showed excellent antipyretic activity as compared to the standard drug in an animal model.

Novel role for mediator complex subunit Srb5/Med18 in termination of transcription.

Mediator complex functions at the recruitment as well as the post-recruitment steps of transcription. Here we provide evidence for a novel role of Mediator in termination of transcription. Mediator subunit Srb5/Med18 cross-links to the 5' and 3' ends of INO1 and CHA1. In srb5(-) cells, recruitment of TATA-binding protein (TBP) and transcription factor IIB (TFIIB) onto the promoter of these genes remained unaffected, but cross-linking of the cleavage-polyadenylation factors Rna15 and Pta1 toward the 3' end of genes was compromised. In these cells, RNA polymerase II accumulated near the 3' end of genes and beyond. Transcription run-on analysis confirmed a transcription readthrough phenotype in the absence of Srb5/Med18. These results strongly suggest that Mediator subunit Srb5/Med18 is required for proper termination of transcription of a subset of genes in budding yeast.

Evidence for a complex of transcription factor IIB with poly(A) polymerase and cleavage factor 1 subunits required for gene looping.

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

Validation of Unani concept of Abadal-i-Adwiya (drug substitution) by physicochemical standardization and hepatoprotective activity of Aristolochia rotunda Linn. and its substitute Curcuma Zedoaria Rosc. in albino Wistar rats.

OBJECTIVES: To validate the concept of abadal-i-adwiya (drug substitution) by evaluation of physicochemical standardization and hepatoprotective activity of Aristolochia rotunda & its substitute, Curcuma Zedoaria in albino Wistar rats. METHODS: Physicochemical standardization by estimation of moisture content, ash values and extractive values were carried out using standard methods. Hepatotoxicity was induced in albino Wistar rats using CCl4 1 mL/kg s. c. on alternate day for 14 days. Group I was served as Plain control and Group II as Negative control. Group III was administered silymarin 50 mg/kg p. o. while Group IV received HAE of A. rotunda 89.64 mg/kg p. o., and Group V was administered HAE of C. Zedoaria 45.73 mg/kg p. o. At the end of the study, serum bilirubin, AST (SGOT), ALT (SGPT) and ALP were estimated. The histopathology of liver was also carried out. RESULTS: The physicochemical parameters of both test drugs viz. moisture content, total ash, acid insoluble ash and water soluble ash were found within normal limit. The total serum bilirubin, direct bilirubin, AST (SGOT), ALT (SGPT) levels were significantly decreased in Test groups A and B when compared to the Negative and Standard controls. The microscopic examination of liver collected from animals of Group IV and Group V revealed significant recovery from hepatic toxicity compared to the Negative control. CONCLUSIONS: The study experimentation has revealed that C. Zedoaria may be used as a substitute for A. rotunda in the treatment of liver diseases. However, the outcome has to be further corroborated with phytochemical evaluation and clinical trials of both the drugs. Furthermore, the concept of drug substitute in Unani system of medicine is also validated in the light of above study.

A Comprehensive Comparison of Clinical Presentation and Outcomes of Kidney Transplant Recipients with COVID-19 during Wave 1 versus Wave 2 at a Tertiary Care Center, India.

Data comparing the clinical spectrum of COVID-19 in kidney transplant recipients (KTRs) during the first and second waves of the pandemic in India is limited. Our single-center retrospective study compared the clinical profile, mortality, and associated risk factors in KTRs with COVID-19 during the 1st wave (1st February 2020 to 31st January 2021) and the second wave (1st March-31st August 2021). 156 KTRs with PCR confirmed SARS-CoV-2 infection treated at a tertiary care hospital in New Delhi during the 1st and the second waves were analyzed. The demographics and baseline transplant characteristics of the patients diagnosed during both waves were comparable. Patients in the second wave reported less frequent hospitalization, though the intensive care unit (ICU) and ventilator requirements were similar. Strategies to modify immunosuppressants such as discontinuation of antinucleoside drugs with or without change in calcineurin inhibitors and the use of steroids were similar during both waves. Overall patient mortality was 27.5%. The demographics and baseline characteristics of survivors and nonsurvivors were comparable. A higher percentage of nonsurvivors presented with breathing difficulty, low SpO2, and altered sensorium. Both wave risk factors for mortality included older age, severe disease, ICU/ventilator requirements, acute kidney injury (AKI) needing dialysis, Chest Computerized Tomographic (CT) scan abnormalities, and higher levels of inflammatory markers particularly D-dimer and interleukin-6 levels. Conclusions. KTRs in both COVID-19 waves had similar demographics and baseline characteristics, while fewer patients during the second wave required hospitalization. The D-dimer and IL-6 levels are directly correlated with mortality.