Transcription termination complex, Rtt103-Rai1-Rat1, regulates sub-telomeric transcripts in Saccharomyces cerevisiae.

Telomeres are terminal structures that define the ends of linear chromosomes. They harbour specialized ribonucleoprotein complexes which play a major role in genome integrity by preventing unscheduled DNA damage repair events. Genes located adjacent to telomere repeat sequences are repressed by a phenomenon called telomere position effect (TPE) via epigenetic silencing. RNA surveillance pathways post-transcriptionally regulate any leaky transcripts arising from the telomeres. Recently, multiple non-coding RNA species originate from telomere ends, namely, TERRA (telomeric repeat-containing RNA), ARRET, sub-telomeric XUTs and sub-telomeric CUTs have been identified. In this study, we report a role for the transcription termination complex (Rtt103-Rai1-Rat1) in regulating the abundance of the sub-telomeric transcripts in a transcription-dependent manner. We show that the Rtt103 mutants have elevated levels of TERRA and other sub-telomeric transcripts that are usually silenced. Our study suggests that Rtt103 potentially recruits the exonuclease, Rat1 in a RNA polymerase II dependent manner to degrade these transcripts and regulate their levels in the cell.

Naringenin-Capped Silver Nanoparticles Amalgamated Gel for the Treatment of Cutaneous Candidiasis.

The current research was aimed to synthesize a phytomolecule, naringenin (NRG)-mediated silver nanoparticles (NRG-SNPs) to study their antifungal potential against Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The NRG-SNPs were synthesized by using NRG as a reducing agent. The synthesis of NRG-SNPs was confirmed by a color change and surface plasmon resonance (SPR) peak at 425 nm. Furthermore, the NRG-SNPs were analyzed for size, PDI, and zeta potential, which were found to be 35 ± 0.21 nm, 0.19 ± 0.03, and 17.73 ± 0.92 mV, respectively. In silico results demonstrated that NRG had a strong affinity towards the sterol 14α-demethylase. The docking with ceramide revealed the skin permeation efficiency of the NRG-SNPs. Next, the NRG-SNPs were loaded into the topical dermal dosage form (NRG-SNPs-TDDF) by formulating a gel using Carbopol Ultrez 10 NF. The MIC50 of NRG solution and TSC-SNPs against C. albicans was found to be 50 µg/mL and 4.8 µg/mL, respectively, significantly (P < 0.05) higher than 0.3625 µg/mL of NRG-SNPs-TDDF. Correspondingly, MIC50 results were calculated against C. glabrata and the results of NRG, TSC-SNPs, NRG-SNPs-TDDF, and miconazole nitrate were found to be 50 µg/mL, 9.6 µg/mL, 0.3625 µg/mL, and 3-µg/mL, respectively. Interestingly, MIC50 of NRG-SNPs-TDDF was significantly (P < 0.05) lower than MIC50 of miconazole nitrate against C. glabrata. The FICI (fractional inhibitory concentration index) value against both the C. albicans and C. glabrata was found to be 0.016 and 0.011, respectively, which indicated the synergistic antifungal activity of NRG-SNPs-TDDF. Thus, NRG-SNPs-TDDF warrants further in depth in vivo study under a set of stringent parameters for translating in to a clinically viable antifungal product.

Nucleolar size regulates nuclear envelope shape in Saccharomyces cerevisiae.

Nuclear shape and size are cell-type specific. Change in nuclear shape is seen during cell division, development and pathology. The nucleus of Saccharomycescerevisiae is spherical in interphase and becomes dumbbell shaped during mitotic division to facilitate the transfer of one nucleus to the daughter cell. Because yeast cells undergo closed mitosis, the nuclear envelope remains intact throughout the cell cycle. The pathways that regulate nuclear shape are not well characterized. The nucleus is organized into various subcompartments, with the nucleolus being the most prominent. We have conducted a candidate-based genetic screen for nuclear shape abnormalities in S. cerevisiae to ask whether the nucleolus influences nuclear shape. We find that increasing nucleolar volume triggers a non-isometric nuclear envelope expansion resulting in an abnormal nuclear envelope shape. We further show that the tethering of rDNA to the nuclear envelope is required for the appearance of these extensions.

The challenge of staying in shape: nuclear size matters.

Cellular organelles have unique morphology and the organelle size to cell size ratio is regulated. Nucleus is one of the most prominent, usually round in shape, organelle of a eukaryotic cell that occupies 8-10% of cellular volume. The shape and size of nucleus is known to undergo remodeling during processes such as cell growth, division and certain stresses. Regulation of protein and lipid distribution at the nuclear envelope is crucial for preserving the nuclear morphology and size. As size and morphology are interlinked, altering one influences the other. In this perspective, we discuss the relationship between size and shape regulation of the nucleus.

Predicting subcellular localization of proteins using protein-protein interaction data.

The knowledge of subcellular localization of proteins can provide useful clues about their functions. The conventional methods to determine the subcellular localization are unable to keep pace with the rate at which the new data is being generated. Thus, though sequence information is available, the localization and function of a number of proteins remains unknown. In this study, we have developed a script that makes use of the physical interactors of a protein and their localization data to predict the subcellular localization. We used the script to predict the localization of yeast proteins for which there is no localization data. Further, we experimentally verified the predicted localization for six arbitrarily chosen proteins and found our predictions to be correct for five of the proteins.

Comparative genomics of nuclear envelope proteins.

BACKGROUND: The nuclear envelope (NE) that encapsulates the nuclear genome is a double lipid bilayer with several integral and peripherally associated proteins. It is a characteristic feature of the eukaryotes and acts as a hub for a number of important nuclear events including transcription, repair, and regulated gene expression. The proteins associated with the nuclear envelope mediate the NE functions and maintain its structural integrity, which is crucial for survival. In spite of the importance of this structure, knowledge of the protein composition of the nuclear envelope and their function, are limited to very few organisms belonging to Opisthokonta and Archaeplastida supergroups. The NE composition is largely unknown in organisms outside these two supergroups. RESULTS: In this study, we have taken a comparative sequence analysis approach to identify the NE proteome that is present across all five eukaryotic supergroups. We identified 22 proteins involved in various nuclear functions to be part of the core NE proteome. The presence of these proteins across eukaryotes, suggests that they are traceable to the Last Eukaryotic Common Ancestor (LECA). Additionally, we also identified the NE proteins that have evolved in a lineage specific manner and those that have been preserved only in a subset of organisms. CONCLUSIONS: Our study identifies the conserved features of the nuclear envelope across eukaryotes and provides insights into the potential composition and the functionalities that were constituents of the LECA NE.

Elevated dosage of Ulp1 disrupts telomeric silencing in Saccharomyces cerevisiae.

Heterochromatin in Saccharomyces cerevisiae is found at the telomeres and silent mating type loci. Many sub-telomeric loci are naturally silenced by this mechanism. In addition, when euchromatic genes are placed proximal to telomeric repeats they are subjected to heritable gene silencing that is referred to as telomere position effect. Establishment and maintenance of TPE is dependent on the assembly of silent information regulator proteins at these loci. Here we show that dosage of SUMO isopeptidase, Ulp1, is important for regulation of TPE. Moderate elevation of Ulp1 reduces silencing of both, the euchromatic gene placed proximal to telomeric repeats and the sub-telomeric genes that are silenced by TPE. We further demonstrate that this loss in silencing is due to reduced recruitment of one of the silent information regulators, Sir3p. We show that SUMO peptidase, Ulp1, regulates telomeric position effect by regulating the recruitment of Sir proteins.

Identification of Components of the SUMOylation Machinery in Candida glabrata: ROLE OF THE DESUMOYLATION PEPTIDASE CgUlp2 IN VIRULENCE.

Regulation of protein function by reversible post-translational modification, SUMOylation, is widely conserved in the eukaryotic kingdom. SUMOylation is essential for cell growth, division, and adaptation to stress in most organisms, including fungi. As these are key factors in determination of fungal virulence, in this study, we have investigated the importance of SUMOylation in the human pathogen, Candida glabrata We identified the enzymes involved in small ubiquitin-like modifier conjugation and show that there is strong conservation between Saccharomyces cerevisiae and C. glabrata We demonstrate that SUMOylation is an essential process and that adaptation to stress involves changes in global SUMOylation in C. glabrata Importantly, loss of the deSUMOylating enzyme CgUlp2 leads to highly reduced small ubiquitin-like modifier protein levels, and impaired growth, sensitivity to multiple stress conditions, reduced adherence to epithelial cells, and poor colonization of specific tissues in mice. Our study thus demonstrates a key role for protein SUMOylation in the life cycle and pathobiology of C. glabrata.

Sumoylation of Sir2 differentially regulates transcriptional silencing in yeast.

Silent information regulator 2 (Sir2), the founding member of the conserved sirtuin family of NAD(+)-dependent histone deacetylase, regulates several physiological processes including genome stability, gene silencing, metabolism and life span in yeast. Within the nucleus, Sir2 is associated with telomere clusters in the nuclear periphery and rDNA in the nucleolus and regulates gene silencing at these genomic sites. How distribution of Sir2 between telomere and rDNA is regulated is not known. Here we show that Sir2 is sumoylated and this modification modulates the intra-nuclear distribution of Sir2. We identify Siz2 as the key SUMO ligase and show that multiple lysines in Sir2 are subject to this sumoylation activity. Mutating K215 alone counteracts the inhibitory effect of Siz2 on telomeric silencing. SUMO modification of Sir2 impairs interaction with Sir4 but not Net1 and, furthermore, SUMO modified Sir2 shows predominant nucleolar localization. Our findings demonstrate that sumoylation of Sir2 modulates distribution between telomeres and rDNA and this is likely to have implications for Sir2 function in other loci as well.

Yeast Nkp2 is required for accurate chromosome segregation and interacts with several components of the central kinetochore.

Kinetochores are macromolecular proteinaceous assemblies that are assembled on centromeres and attach chromosomes to the spindle fibres and regulate the accurate transmission of genetic material to daughter cells. Multiple protein sub-complexes within this supramolecular assembly are hierarchically assembled and contribute to the different aspects of kinetochore function. In this work we show that one of the components of the Saccharomyces cerevisiae kinetochore, Nkp2, plays an important role in ensuring accurate segregation of chromosomes. Although this protein is not conserved in higher organisms, we show that it interacts with highly conserved components of the kinetochore genetically and regulates chromosome segregation. We show that in kinetochore mutants like ctf19 and mcm21 the protein is mislocalized. Furthermore, removal of Nkp2 in these mutants restores normal levels of segregation.

The nuclear pore protein NUP98 impedes LTR-driven basal gene expression of HIV-1, viral propagation, and infectivity.

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.

AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila.

Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes.

The SUMO E3 ligase Siz2 exerts a locus-dependent effect on gene silencing in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the two silent mating-type loci and subtelomeric regions are subjected to a well-characterized form of gene silencing. Establishment of silencing involves the formation of a distinct chromatin state that is refractory to transcription. This structure is established by the action of silent information regulator proteins (Sir2, Sir3, and Sir4) that bind to nucleosomes and initiate the deacetylation of multiple lysine residues in histones H3 and H4. Sir2 protein is a conserved histone deacetylase that is critical for mating-type and telomeric silencing, as well as a Sir3/4-independent form of silencing observed within the ribosomal DNA (rDNA) repeat locus. We report here that sumoylation plays an important role in regulating gene silencing. We show that increased dosage of SIZ2, a SUMO (small ubiquitin-related modifier) ligase, is antagonistic to gene silencing and that this effect is enhanced by mutation of ESC1, whose product is involved in tethering telomeres to the nuclear periphery. We present evidence indicating that an elevated SIZ2 dosage causes reduced binding of Sir2 protein to telomeres. These data support the idea that sumoylation of specific substrates at the nuclear periphery regulates the availability of Sir2 protein at telomeres.

Functional diversification of yeast telomere associated protein, Rif1, in higher eukaryotes.

BACKGROUND: Telomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity. RESULTS: We have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci. CONCLUSIONS: Telomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.

An adaptable live-cell imaging protocol to analyze organelle morphology in Saccharomyces cerevisiae.

The protocol describes semiautomated live cell imaging in budding yeast. A key feature of the protocol is immobilizing cells in a culture dish, which allows for longer imaging times, changing culture media, or drug treatments. We describe steps for image acquisition and deconvolution, followed by manual analysis of quantifiable parameters to represent morphological changes in nuclear shape. We compare wild type with ssf1Δ, which is known to alter nuclear morphology. The protocol can be adapted to other organelles and processes. For complete details on the use and execution of this profile, please refer to Male et al., 2020, Deolal et al. (2021).

RNA-Mediated Regulation of Meiosis in Budding Yeast.

Cells change their physiological state in response to environmental cues. In the absence of nutrients, unicellular fungi such as budding yeast exit mitotic proliferation and enter the meiotic cycle, leading to the production of haploid cells that are encased within spore walls. These cell state transitions are orchestrated in a developmentally coordinated manner. Execution of the meiotic cell cycle program in budding yeast, Saccharomyces cerevisiae, is regulated by the key transcription factor, Ime1. Recent developments have uncovered the role of non-coding RNA in the regulation of Ime1 and meiosis. In this review, we summarize the role of ncRNA-mediated and RNA homeostasis-based processes in the regulation of meiosis in Saccharomyces cerevisiae.

Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae.

A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localization studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.

Regulation of diverse nuclear shapes: pathways working independently, together.

Membrane-bound organelles provide physical and functional compartmentalization of biological processes in eukaryotic cells. The characteristic shape and internal organization of these organelles is determined by a combination of multiple internal and external factors. The maintenance of the shape of nucleus, which houses the genetic material within a double membrane bilayer, is crucial for a seamless spatio-temporal control over nuclear and cellular functions. Dynamic morphological changes in the shape of nucleus facilitate various biological processes. Chromatin packaging, nuclear and cytosolic protein organization, and nuclear membrane lipid homeostasis are critical determinants of overall nuclear morphology. As such, a multitude of molecular players and pathways act together to regulate the nuclear shape. Here, we review the known mechanisms governing nuclear shape in various unicellular and multicellular organisms, including the non-spherical nuclei and non-lamin-related structural determinants. The review also touches upon cellular consequences of aberrant nuclear morphologies.

Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.

SUMOylation in fungi: A potential target for intervention.

SUMOylation is a post-translational, reversible modification process which occurs in eukaryotes. Small Ubiquitin like MOdifier or (SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins to modify the target protein function. In pathogenic fungi, SUMO has been identified and preliminary studies indicate its importance either for survival and/or for virulence. In this review we provide an overview of the current state of knowledge of SUMOylation in fungi and the effects on pathogenesis. Subsequently we identify the orthologs of the SUMOylation pathway components across fungi. We also show the level of conservation of the proteins involved and identify the similarities/differences in the orthologs across fungi and the human and plant hosts to identify potential targets of intervention.