The RNA polymerase gene specificity factor sigma 54 is required for homogeneous non-planktonic growth of uropathogenic Escherichia coli

The canonical function of a bacterial sigma factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the sigma 54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the sigma 54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. The genes that rely upon sigma 54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by sigma 54, is not yet exhaustive. By comparing the non-planktonic growth properties of prototypical enteropathogenic, uropathogenic and non-pathogenic Escherichia coli strains devoid of sigma 54, we uncovered sigma 54 as a determinant of homogenous non-planktonic growth specifically in the uropathogenic strain. Notably, bacteria devoid of individual activator ATPases of the sigma 54-RNAP do not phenocopy the sigma 54 mutant strain. It seems that sigma 54's role as a determinant of homogenous non-planktonic growth represents a putative non-canonical function of sigma 54 in regulating genetic information flow.

RBBP6 activates the pre-mRNA 3′-end processing machinery in humans

3′-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3′-endonuclease activity of human CPSF with purified proteins. This requires the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

Abstract P501: Comprehensive Analysis Of Alternative Polyadenylation In Human Left Ventricle Failure By Using The Novel Algorithm: Polya-miner

Background: Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3′ untranslated regions (3’UTR) lengths. APA plays an important role in different biological processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the left ventricle (LV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the LV failure in humans compared to healthy LV. Methods and Results: We used Poly(A)-ClickSeq RNA sequencing and PolyA-miner algorithm to measure the global patterns of APA in healthy and failing human LV specimens. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 129 genes with a significant shift of cleavage site usage in failing LV compared to healthy LV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in LV failure regulate many pathways important for the progression of LV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in LV failure compared to healthy LV specimens. Conclusions: Our results provide genome-wide, polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure in humans. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in LV failure.

Cleavage and Polyadenylation Specificity Factor 6 Is Required for Efficient HIV-1 Latency Reversal

CPSF6 is a cellular factor that regulates cleavage and polyadenylation of mRNAs and participates in HIV-1 infection by facilitating targeting of preintegration complexes to the chromatin. Our observations reveal a second role of CPSF6 in the HIV-1 life cycle that involves regulation of viral transcription through controlling the stability of protein phosphatase 2A, which in turn regulates the phosphorylation/dephosphorylation status of critical residues in CDK9 and Pol II.

A large lipoprotein mediates target specificity for T6SS-dependent killing

Interbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a specificity factor in Vibrio species that is used to target specific competitors for elimination. Here, we describe this specificity factor, which is associated with the broadly-distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which use the T6SS to compete for colonization sites in their squid host. We demonstrate that a large lipoprotein (TasL) allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly-described specificity factor has the potential to impact microbiome structure within diverse hosts.

Characteristics of Viburnum species seeds

Viburnums are of economic importance, are an important component of forests, they are used in landscaping, have medicinal and nutritional value. The purpose of the study is a comparative analysis of characteristics of seeds of 4 viburnum species in the conditions of the Mari El Republic. The authors study the pyrenes collected in 2018-2020 from the plants grown in the exposition "Fruticetum" of the Botanical Garden-Institute of VSUT (Yoshkar-Ola). The mass of 1000 seeds was determined according to GOST 13056.4-67, the sizes of 30 seeds were measured with a caliper. It was found that the studied viburnums form seeds of typical size and weight. Viburnum lentago had the largest seeds, Viburnum lantana had the smallest ones. A significant influence of the species specificity factor on the weight of seeds was revealed, while no significant influence of the factor of weather conditions of different years on the seed indicators was established. The weight of the seeds was very closely positively correlated with their length and significantly with their thickness. The thickness of the seeds was characterized by the highest level of variability, negatively correlated with their width and positively with their length. During the 3 years of the study, the heaviest seeds in most species were formed in 2018.

Chromatin Regulator SPEN/SHARP in X Inactivation and Disease

Enzymes, such as histone methyltransferases and demethylases, histone acetyltransferases and deacetylases, and DNA methyltransferases are known as epigenetic modifiers that are often implicated in tumorigenesis and disease. One of the best-studied chromatin-based mechanism is X chromosome inactivation (XCI), a process that establishes facultative heterochromatin on only one X chromosome in females and establishes the right dosage of gene expression. The specificity factor for this process is the long non-coding RNA Xinactivespecifictranscript (Xist), which is upregulated from one X chromosome in female cells. Subsequently, Xist is bound by the corepressor SHARP/SPEN, recruiting and/or activating histone deacetylases (HDACs), leading to the loss of active chromatin marks such as H3K27ac. In addition, polycomb complexes PRC1 and PRC2 establish wide-spread accumulation of H3K27me3 and H2AK119ub1 chromatin marks. The lack of active marks and establishment of repressive marks set the stage for DNA methyltransferases (DNMTs) to stably silence the X chromosome. Here, we will review the recent advances in understanding the molecular mechanisms of how heterochromatin formation is established and put this into the context of carcinogenesis and disease.

Cytoplasmic CPSF6 Regulates HIV-1 Capsid Trafficking and Infection in a Cyclophilin A-Dependent Manner

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages. IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4+ T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis.

HIV-1 cores retain their integrity until minutes before uncoating in the nucleus

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

SummaryCells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. The binding of the TCR-specific repair factor CSB triggers DNA damage-induced ubiquitylation of RNA polymerase II (RNAPII) at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. However, how the CRL4CSA ligase is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation. Furthermore, drug-genetic interaction screening reveals an unanticipated compensatory TCR pathway in which ELOF1 together with known factors DOT1L and HIRA protect CSB-deficient cells from collisions between transcription and replication machineries. Our study provides a genetic framework of the transcription stress response and reveals key insights into the molecular mechanism of TCR.