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.

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.

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.

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.

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.

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.

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.

Mortality and Clinical Outcomes among Patients with COVID-19 and Diabetes.

Background Diabetes mellitus (DM) is a decisive risk factor for severe illness in coronavirus disease 2019 (COVID-19). India is home to a large number of people with DM, and many of them were infected with COVID-19. It is critical to understand the impact of DM on mortality and other clinical outcomes of COVID-19 infection from this region. Aims The primary objective of our study was to analyze the mortality rate in people with DM infected with COVID-19. The secondary objectives were to assess the effect of various comorbidities on mortality and study the impact of DM on other clinical outcomes. Methods This is a retrospective study of COVID-19 infected patients admitted to a tertiary care hospital in north India in the early phase of the pandemic. Results Of the 1211 cases admitted, 19 were excluded because of incomplete data, and 1192 cases were finally considered for analysis. DM constituted 26.8% of total patients. The overall mortality rate was 6.1%, and the rate was 10.7% in the presence of diabetes (p < 0.01, OR 2.55). In univariate analysis, increased age, chronic kidney disease (CKD), coronary artery disease (CAD), stroke, and cancer were associated with mortality. On multiple logistic regression, the independent predictors of mortality were CAD, CKD, and cancer. Breathlessness and low SpO2 at presentation, extensive involvement in CXR, and elevated ANC/ALC ratio were also significantly associated with mortality. Conclusions The presence of comorbidities such as DM, hypertension, CAD, CKD, and cancer strongly predict the risk of mortality in COVID-19 infection. Early triaging and aggressive therapy of patients with these comorbidities can optimize clinical outcomes.

Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron.

An evolutionarily conserved feature of introns is their ability to enhance expression of genes that harbor them. Introns have been shown to regulate gene expression at the transcription and post-transcription level. The general perception is that a promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression when in proximity to the terminator. We inserted ACT1 intron into different positions within IMD4 and INO1 genes. Transcription Run-On (TRO) analysis revealed that the transcription of both IMD4 and INO1 was maximal in constructs with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. However, activation was partially restored when the intron was placed close to the terminator. We previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through gene looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron also enhances promoter directionality and results in compact gene architecture with the promoter and terminator regions in close physical proximity. Furthermore, we show that both the promoter and terminator-proximal introns facilitate assembly or stabilization of the preinitiation complex (PIC) on the promoter. On the basis of these findings, we propose that proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron, and the terminator-proximal intron enhances transcription by affecting both the assembly of preinitiation complex and promoter directionality.

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.

Critical Involvement of TFIIB in Viral Pathogenesis.

Viral infections and the harm they cause to their host are a perpetual threat to living organisms. Pathogenesis and subsequent spread of infection requires replication of the viral genome and expression of structural and non-structural proteins of the virus. Generally, viruses use transcription and translation machinery of the host cell to achieve this objective. The viral genome encodes transcriptional regulators that alter the expression of viral and host genes by manipulating initiation and termination steps of transcription. The regulation of the initiation step is often through interactions of viral factors with gene specific factors as well as general transcription factors (GTFs). Among the GTFs, TFIIB (Transcription Factor IIB) is a frequent target during viral pathogenesis. TFIIB is utilized by a plethora of viruses including human immunodeficiency virus, herpes simplex virus, vaccinia virus, Thogoto virus, hepatitis virus, Epstein-Barr virus and gammaherpesviruses to alter gene expression. A number of viral transcriptional regulators exhibit a direct interaction with host TFIIB in order to accomplish expression of their genes and to repress host transcription. Some viruses have evolved proteins with a three-dimensional structure very similar to TFIIB, demonstrating the importance of TFIIB for viral persistence. Upon viral infection, host transcription is selectively altered with viral transcription benefitting. The nature of viral utilization of TFIIB for expression of its own genes, along with selective repression of host antiviral genes and downregulation of general host transcription, makes TFIIB a potential candidate for antiviral therapies.

Gene Architecture Facilitates Intron-Mediated Enhancement of Transcription.

Introns impact several vital aspects of eukaryotic organisms like proteomic plasticity, genomic stability, stress response and gene expression. A role for introns in the regulation of gene expression at the level of transcription has been known for more than thirty years. The molecular basis underlying the phenomenon, however, is still not entirely clear. An important clue came from studies performed in budding yeast that indicate that the presence of an intron within a gene results in formation of a multi-looped gene architecture. When looping is defective, these interactions are abolished, and there is no enhancement of transcription despite normal splicing. In this review, we highlight several potential mechanisms through which looping interactions may enhance transcription. The promoter-5' splice site interaction can facilitate initiation of transcription, the terminator-3' splice site interaction can enable efficient termination of transcription, while the promoter-terminator interaction can enhance promoter directionality and expedite reinitiation of transcription. Like yeast, mammalian genes also exhibit an intragenic interaction of the promoter with the gene body, especially exons. Such promoter-exon interactions may be responsible for splicing-dependent transcriptional regulation. Thus, the splicing-facilitated changes in gene architecture may play a critical role in regulation of transcription in yeast as well as in higher eukaryotes.

Crosstalk of promoter and terminator during RNA polymerase II transcription cycle.

The transcription cycle of RNAPII is comprised of three consecutive steps; initiation, elongation and termination. It has been assumed that the initiation and termination steps occur in spatial isolation, essentially as independent events. A growing body of evidence, however, has challenged this dogma. First, factors involved in initiation and termination exhibit both a genetic and a physical interaction during transcription. Second, the initiation and termination factors have been found to occupy both ends of a transcribing gene. Third, physical interaction of initiation and termination factors occupying distal ends of a gene sometime results in the entire terminator region of a genes looping back and contact its cognate promoter, thereby forming a looped gene architecture during transcription. A logical interpretation of these findings is that the initiation and termination steps of transcription do not occur in isolation. There is extensive communication of factors occupying promoter and terminator ends of a gene during transcription cycle. This review entails a discussion of the promoter-terminator crosstalk and its implication in the context of transcription.

Safety of day-case endovascular interventions for peripheral arterial disease in a rural, underserved area.

BACKGROUND: We aimed to investigate the safety of endovascular procedures undertaken in a single outpatient center located in a rural, underserved area. Endovascular procedures for Peripheral Arterial Disease (PAD) have become increasingly common in outpatient settings; their safety is yet to be determined in a rural, underserved area with no stand-by vascular surgeon on site. METHODS: We undertook a retrospective case review of endovascular procedures for the investigation and management of lower extremity PAD between December 2012 and August 2015. Patients were classified by Rutherford score, degree of stenosis and length of lesions. Complications were major (requiring hospitalization) or minor, including perforation, distal embolization, hematoma, and allergic reactions, which could be treated immediately in the catheterization laboratory with no sequelae. Patients were monitored in the facility and followed up using clinical, biochemical and radiological parameters at 24 h and 1 month. RESULTS: A total of 692 patients underwent endovascular procedures for the investigation and/or treatment of PAD, of which 608 were interventional. Of these patients, 10.20% experienced procedural complications, of which 0.66% were classified as major, including wire retention and retroperitoneal hemorrhage. In total, 99.34% were discharged safely on the same day as the procedure. No adverse events were reported at follow up. CONCLUSION: Endovascular procedures for PAD can be performed safely in a rural outpatient setting with low complication rates. Most complications are minor and do not require hospitalization. Outpatient procedures for PAD are safe and may widen access to specialist procedures in areas of socio-economic deprivation.

'Ilaj bi'l-Tadbir (regimenal therapy): a core mode of Unani treatment.

Since antiquity, the Unani system of medicine has been participating in health care system. Usually, four modes of treatment viz. regimenal therapy, dietotherapy, pharmacotherapy and surgery are applied for the treatment of diseases. Regimenal therapy is an important mode in which the morbid matter present in the body is either dispersed/excreted or its unnecessary production is blocked or its flow is restricted and the diseases are cured by natural healer of the body, consequently bring back the humoural stability. Nearly 30 regimens have been mentioned in classical Unani literature. Commonest regimenal procedures such as fasd (venesection/phlebotomy), hijama (cupping), ta'liq al-'alaq (hirudotherapy/leech therapy), ishal (purgation), qay' (emesis), idrar-i-bawl (diuresis), huqna (enema), ta'riq (diaphoresis), riyadat (exercise), dalk (massage), hammam (bathing), tadhin (oiling), natul (irrigation), sakub (douching/spraying), inkibab (steam/vapour application), takmid (fomentation) etc. are usually applied for the management of various ailments. These regimenal procedures are completely based on holistic approach and are potential but needs to be explored scientifically. This review outlines the therapeutic applications of various regimens of regimenal therapy used in Unani medicine.

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.

Genomewide Analysis of Clp1 Function in Transcription in Budding Yeast.

In budding yeast, the 3' end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent termination of transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled termination of transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled termination of RNAPII transcription and in enhancing promoter directionality in budding yeast.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach.

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3' end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3' end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

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.