BackgroundOver the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.ResultsWe exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.ConclusionsThe simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-959) contains supplementary material, which is available to authorized users.

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA Gln gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA Gln , the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA Gln blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes and various other chromatin proteins. Chromatin structure varies along the chromosome, and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (transcriptionally repressed). Mechanisms that demarcate heterochromatin domains from euchromatin are actively being investigated (4,16,60), and several models have been proposed. For example, the boundary at telomeric heterochromatin in Saccharomyces cerevisiae is maintained by a balance between the opposing activities of Sir2 and Sas2, which deacetylate and acetylate histone H4-K16, respectively (28, 54). The right boundary at the cryptic matingtype locus HML is established by directional initiation and spreading of silencing inward, toward the mating-type genes, from the HML I silencer (3). In contrast, HMR silencing spreads bidirectionally from the silencers and is limited by sequences termed boundary elements or "silencing barriers" (14). The molecular mechanism of barrier activity is not well understood. Existing models include physical hindrance to the spreading of silencing proteins by bound multiprotein complexes, tethering to nuclear structural elements such as the scaffold or nuclear envelope, nucleosome exclusion, or recruitment of euchromatinizing/antisilencing factors that may modify chromatin at the barrier such that it is unfavorable for spreading of heterochromatin (6,15,25,39,40).The rDNA (RDN1) locus, which has ϳ150 copies of a tandemly repeated sequence encoding rR...

Genetic information in cells is encrypted in DNA molecules forming chromosomes of varying sizes. Accurate replication and partitioning of chromosomes in the crowded cellular milieu is a complex process involving duplication, folding and movement. Longer chromosomes may be more susceptible to mis-segregation or DNA damage and there may exist specialized physiological mechanisms preventing this. Here, we present genetic evidence for such a mechanism which depends on Mms21/Nse2 mediated sumoylation and topoisomerase-1 (Top1) for maintaining stability of longer chromosomes. While mutations inactivating Top1 or the SUMO ligase activity of Mms21 (mms21sl) individually destabilized yeast artificial chromosomes (YACs) to a modest extent, the mms21sl top1 double mutant exhibited a synthetic-sick phenotype, and showed preferential destabilization of the longer chromosome relative to shorter chromosomes. In contrast, an smc6-56 top1 mutant defective in Smc6, another subunit of the Smc5/6 complex, of which Mms21 is a component, did not show such a preferential enhancement in frequency of loss of the longer YAC, indicating that this defect may be specific to the deficiency in SUMO ligase activity of Mms21 in the mms21sl top1 mutants. In addition, mms21sl top1 double mutants harboring a longer fusion derivative of natural yeast chromosomes IV and XII displayed reduced viability, consistent with enhanced chromosome instability, relative to single mutants or the double mutant having the natural (shorter) non-fused chromosomes. Our findings reveal a functional interplay between Mms21 and Top1 in maintenance of longer chromosomes, and suggest that lack of sumoylation of Mms21 targets coupled with Top1 deficiency is a crucial requirement for accurate inheritance of longer chromosomes.

18NOT1 is the scaffold of the CCR4-NOT complex, a highly conserved multi-protein complex that 19 regulates gene expression in eukaryotes. As opposed to most eukaryotes in which NO1 is 20 encoded by a single gene, malaria parasites, Plasmodium falciparum, carry two NOT1 21 paralogues, PfNOT1.1 and PfNOT1.2. Here we showed that the two PfNOT1 proteins function as 22 3 38 possess an additional copy of PfNOT1 in the parasite. Here we described antagonistic regulatory 39 functions of two PfNOT1 paralogues in gene expression during the 48-hour intraerythrocytic 40 developmental cycle. We also reported that their regulatory functions are predominantly post-41 transcriptional and proposed a model in which distinct PfCCR4-NOT complexes defined by 42 mutually exclusive PfNOT1 scaffolds differentially regulate PfCAF1 function in mRNA decay. This 43 study highlights the importance of post-transcriptional regulation in P. falciparum and provides 44 novel insights into mechanisms of gene regulation in this organism. The unique presence of two 45 PfNOT1 paralogues may also open avenues for the development of new drug targets for anti-46 malarial control. 47 4 58 expression [4]. Hence, gene expression is highly regulated across the IDC at multiple levels, 59 ranging from epigenetic regulation, transcription and post-transcriptional regulation. 60 While the basic mechanism of eukaryotic transcription and general transcription components 61 are conserved in P. falciparum [5, 6], most TBP-associated factors (TAF) are absent [6] and only 62 a third of the expected number of canonical eukaryotic transcription activating proteins (TAP) 63 were predicted in the parasite genome [7]. The presence of 27 Apicomplexan-specific 64 transcription factors, ApiAP2, may compensate for the reduction in the number of conventional 65 transcription components [8, 9]. These 27 ApiAP2 proteins display distinct expression profiles 66 covering the entire IDC [8] and their recognition motifs are located in the promoter regions of 67 most genes whose expression profiles are positively correlated to expression of the 68 corresponding ApiAP2 gene [9]. In addition to transcriptional initiation, there is mounting 69 evidence that multiple processes of post-transcriptional regulation are also critical for 70 regulation of mRNA abundance through the P. falciparum IDC [10-12]. This is supported by the 71 abundant presence RNA-binding proteins [13, 14] and RNA degradation components [15] in the 72 P. falciparum genome. The includePfALBA1 [16], PfCAF1 [17] in P. falciparum and PyCCR4 [18], 73 PyALBA4 [19] in rodent-specific P.yoelii all of which functions were directly implicated in 74 regulation of transcript levels. Moreover, variability of mRNA half-lives across the IDC observed 75 by several experimental approaches also suggested a significant contribution of mRNA decay in 76 regulation of gene expression in malaria parasites [20]. Taken together, both transcriptional 77 regulation and post-transcriptional regulation play critical roles in modulating transcr...

Replication of linear chromosomes is facilitated by firing of multiple replication origins that ensures timely duplication of the entire chromosome. The Smc5/6 complex is thought to play an important role in replication by its involvement in the restart of collapsed replication forks. Here, we present genetic evidence for functional interaction between replication origin distribution and two subunits of the Smc5/6 complex, Smc6 and Mms21, as well as Top1. An artificial chromosome that has a long arm having low origin density (5ori∆YAC) is relatively unstable compared to the YAC having normal origin distribution in wild-type cells, but is partially stabilized in smc6-56 and top1∆ mutants. While a SUMO-ligase-deficient mutant of Mms21 does not affect stability of the 5ori∆YAC by itself, in combination with top1∆, the 5ori∆YAC is destabilized as evidenced by increased chromosome loss frequency in the mms21∆sl top1∆ double mutant. Likewise, the smc6-56 top1∆ double mutant also exhibits enhanced destabilization of the 5ori∆YAC compared to either single mutant. Such an increase in chromosome loss is not observed for a similar YAC that retains the original replication origins and normal origin distribution on the long arm, in either double mutant having the mms21∆sl or smc6-56 mutations in combination with top1∆. Our findings reveal a requirement for the Smc5/6 complex, including Mms21/Nse2 mediated sumoylation, and topoisomerase-1 (Top1), for maintaining stability of a chromosome having low origin density and suggest a functional cooperation between the Smc5/6 complex and Top1 in maintenance of topologically challenged chromosomes prone to replication fork collapse or accumulation of torsional stress.

Advances in our understanding of complex COVID-19 pandemic would allow us to effectively eliminate and eradicate SARS-COV2 virus. Although tremendous amount of research devoted to the robustness across its biology, diagnostics, vaccines and treatment has exploded in the past two years. However, still science do not have robust answers for causes, for example (i) What are the reasons of non-uniform global distribution of COVID-19? (ii) Why the United States, India, and Brazil, are the first-three most affected nations?, (iii) How did Bhutan, a nation sharing a boundary with China manage nearly 0.34% infections and 3 deaths from COVID-19? Nonetheless, the biomass bistribution of biosphere report suggest more than 1550-fold larger microbial biomass involving bacteria, fungi, archaea, protists and viruses is exist in comparision to all global human population in the biosphere. The rich microbiota act a first line of defence to invade pathogens and affect us both through the environment and microbiome. Unfortunately, a role of pathogen-transmission factors viz. implicit factors (competitive microflora) is still under represented. This study is an attempt from a gold standard correlation methodology using a large pesticide use global data. The non-specific pesticides kill both pests as well as protective microbiota, resulting a loss in rich biodiversity and allow easy pathogen entry to human. Entire predictions were found consistent with the recently observed evidences. These insights enhanced scientific ability to interrogate viral epidemiology and recommended to limit pesticide use for future pandemic prevention.