Spatiotemporal Analysis of DNA Integration during Natural Transformation Reveals a Mode of Nongenetic Inheritance in Bacteria.

Natural transformation (NT) is a major mechanism of horizontal gene transfer in microbial species that promotes the spread of antibiotic-resistance determinants and virulence factors. Here, we develop a cell biological approach to characterize the spatiotemporal dynamics of homologous recombination during NT in Vibrio cholerae. Our results directly demonstrate (1) that transforming DNA efficiently integrates into the genome as single-stranded DNA, (2) that the resulting heteroduplexes are resolved by chromosome replication and segregation, and (3) that integrated DNA is rapidly expressed prior to cell division. We show that the combination of these properties results in the nongenetic transfer of gene products within transformed populations, which can support phenotypic inheritance of antibiotic resistance in both V. cholerae and Streptococcus pneumoniae. Thus, beyond the genetic acquisition of novel DNA sequences, NT can also promote the nongenetic inheritance of traits during this conserved mechanism of horizontal gene transfer.

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

Mechanisms of DNA Uptake by Naturally Competent Bacteria.

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

SoxC transcription factors shape the epigenetic landscape to establish competence for sensory differentiation in the mammalian organ of Corti.

The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.

Enhancer, transcriptional, and cell fate plasticity precedes intestinal determination during endoderm development.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

Setting the clock of neural progenitor cells during mammalian corticogenesis.

Radial glial cells (RGCs) as primary neural stem cells in the developing mammalian cortex give rise to diverse types of neurons and glial cells according to sophisticated developmental programs with remarkable spatiotemporal precision. Recent studies suggest that regulation of the temporal competence of RGCs is a key mechanism for the highly conserved and predictable development of the cerebral cortex. Various types of epigenetic regulations, such as DNA methylation, histone modifications, and 3D chromatin architecture, play a key role in shaping the gene expression pattern of RGCs. In addition, epitranscriptomic modifications regulate temporal pre-patterning of RGCs by affecting the turnover rate and function of cell-type-specific transcripts. In this review, we summarize epigenetic and epitranscriptomic regulatory mechanisms that control the temporal competence of RGCs during mammalian corticogenesis. Furthermore, we discuss various developmental elements that also dynamically regulate the temporal competence of RGCs, including biochemical reaction speed, local environmental changes, and subcellular organelle remodeling. Finally, we discuss the underlying mechanisms that regulate the interspecies developmental tempo contributing to human-specific features of brain development.

An Extracellular, Ca2+-Activated Nuclease (EcnA) Mediates Transformation in a Naturally Competent Archaeon.

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca2+-activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion.

WNV and SLEV coinfection in avian and mosquito hosts: impact on viremia, antibody responses, and vector competence.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related flaviviruses that can cause encephalitis in humans and related diseases in animals. In nature, both are transmitted by Culex, with wild birds, including jays, sparrows, and robins, serving as vertebrate hosts. WNV and SLEV circulate in the same environments and have recently caused concurrent disease outbreaks in humans. The extent that coinfection of mosquitoes or birds may alter transmission dynamics, however, is not well characterized. We therefore sought to determine if coinfection alters infection kinetics and virus levels in birds and infection rates in mosquitoes. Accordingly, American robins (Turdus migratorius), two species of mosquitoes, and vertebrate and invertebrate cells were infected with WNV and/or SLEV to assess how simultaneous exposure may alter infection outcomes. There was variable impact of coinfection in vertebrate cells, with some evidence that SLEV can suppress WNV replication. However, robins had comparable viremia and antibody responses regardless of coinfection. Conversely, in Culex cells and mosquitoes, we saw a minimal impact of simultaneous exposure to both viruses on replication, with comparable infection, dissemination, and transmission rates in singly infected and coinfected mosquitoes. Importantly, while WNV and SLEV levels in coinfected mosquito midguts were positively correlated, we saw no correlation between them in salivary glands and saliva. These results reveal that while coinfection can occur in both avian and mosquito hosts, the viruses minimally impact one another. The potential for coinfection to alter virus population structure or the likelihood of rare genotypes emerging remains unknown.IMPORTANCEWest Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related viruses that are transmitted by the same mosquitoes and infect the same birds in nature. Both viruses circulate in the same regions and have caused concurrent outbreaks in humans. It is possible that mosquitoes, birds, and/or humans could be infected with both WNV and SLEV simultaneously, as has been observed with Zika, chikungunya, and dengue viruses. To study the impact of coinfection, we experimentally infected vertebrate and invertebrate cells, American robins, and two Culex species with WNV and/or SLEV. Robins were efficiently coinfected, with no impact of coinfection on virus levels or immune response. Similarly, in mosquitoes, coinfection did not impact infection rates, and mosquitoes could transmit both WNV and SLEV together. These results reveal that WNV and SLEV coinfection in birds and mosquitoes can occur in nature, which may impact public health and human disease risk.

Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Histone H2A variant H2A.B is enriched in transcriptionally active and replicating HSV-1 lytic chromatin.

Herpes simplex virus 1 (HSV-1) transcription is restricted in latently infected neurons and the genomes are in mostly silenced chromatin, whereas all viral genes are transcribed in lytically infected cells, in which the genomes are dynamically chromatinized. Epigenetic regulation modulates HSV-1 transcription during lytic, latent, and reactivating infections but the precise mechanisms are not fully defined. Nucleosomes are dynamic: they slide, breathe, assemble, and disassemble. We and others have proposed that the most dynamic HSV-1 chromatin is transcriptionally competent, whereas the least dynamic is silenced. However, the mechanisms yielding the unusually dynamic viral chromatin remain unknown. Histone variants affect nucleosome dynamics. The dynamics of H2A, H2A.X, and macroH2A were enhanced in infected cells, whereas those of H2A.B were uniquely decreased. We constructed stably transduced cells expressing tagged histone H2A, H2A.B, macroH2A, or H2B, which assembles the H2A/H2B nucleosome dimers with all H2A variants. All H2A variants, as well as ectopic and endogenous H2B were assembled into HSV-1 chromatin evenly throughout the genome but canonical H2A was relatively depleted whereas H2A.B was enriched, particularly in the most dynamic viral chromatin. When viral transcription and DNA replication were restricted, H2A.B became as depleted from the viral chromatin through the entire genome as H2A. We propose that lytic HSV-1 nucleosomes are enriched in the dynamic variant H2A.B/H2B dimers to promote HSV-1 chromatin dynamics and transcriptional competency and conclude that the dynamics of HSV-1 chromatin are determined in part by the H2A variants. IMPORTANCE: Herpes simplex virus 1 (HSV-1) transcription is epigenetically regulated during latent and lytic infections, and epigenetic inhibitors have been proposed as potential antiviral drugs to modulate latency and reactivation. However, the detailed epigenetic mechanisms of regulation of HSV-1 transcription have not been fully characterized and may differ from those regulating cellular transcription. Whereas lytic HSV-1 chromatin is unusually dynamic, latent silenced HSV-1 chromatin is not. The mechanisms resulting in the unique dynamics of the lytic chromatin remain unknown. Here we identify the enrichment of the highly dynamic histone 2A variant H2A in the most dynamic viral chromatin, which provides a mechanistic understanding of its unique dynamics. Future work to identify the mechanisms of enrichment in H2A.B on the viral chromatin may identify novel druggable epigenetic regulators that modulate HSV-1 latency and reactivation.

The influence of self-esteem on interpersonal and competence evaluations: electrophysiological evidence from an ERP study.

Using event-related potentials, this study examined how self-esteem affects neural responses to competence (interpersonal) feedback when the need for relatedness (competence) is thwarted or met. Participants with low and high self-esteem acted as advisors who selected one of two options for a putative advisee. Subsequently, they passively observed the advisee, accepted, or rejected their advice (i.e. interpersonal feedback) and received correct or incorrect outcomes (i.e. competence feedback). When interpersonal feedback was followed by competence feedback, high self-esteem participants showed a smaller P3 following incorrect than correct outcomes, irrespective of whether the advice had been accepted or rejected. However, low self-esteem participants showed this P3 effect only when the advice was rejected, and the P3 difference disappeared when the advice was accepted. When competence feedback was followed by interpersonal feedback, both low self-esteem and high self-esteem individuals showed a larger P2 for rejection than for acceptance and a larger late potential component for incorrect than correct outcomes. These findings suggest that when interpersonal feedback is followed by competence feedback, low self-esteem and high self-esteem individuals have a desire for self-positivity. When competence feedback is followed by interpersonal feedback, they may have motives for self-change. Our findings shed light on the motivational mechanisms for self-esteem and feedback.

Achievement Goals: A Social Influence Cycle.

Achievement goals have been defined as the purpose of competence-relevant behavior. In this respect they connect one of the basic human needs, i.e., competence, to one of society's core values, i.e., achievement. We propose to look at achievement goals through the lens of social influence. We review both the influence that cultural, structural, and contextual factors have on achievement goal endorsement and the influence that endorsing achievement goals allows people to have within their social space. The review allows us to propose a circular model of the influence on and of achievement goals: The culture, social structures, and contexts that are typical of a certain society shape the specific environments in which individuals develop their achievement goals, which in turn has an influence on the expression and circulation of these achievement goals into society, in a social influence cycle.

ComI inhibits transformation in Bacillus subtilis by selectively killing competent cells.

Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, Bacillus subtilis, has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is expressed at higher levels in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins, and permeabilization activity is concentration-dependent. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation. IMPORTANCE: Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of Bacillus subtilis into the "competent" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.

Signal recognition particle RNA is critical for genetic competence and virulence of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) causes a wide range of important human infectious diseases, including pneumonia, pneumonia-derived sepsis, otitis media, and meningitis. Pneumococcus produces numerous secreted proteins that are critical for normal physiology and pathogenesis. The membrane targeting and translocation of these secreted proteins are partly mediated by the signal recognition particle (SRP) complex, which consists of 4.5S small cytoplasmic RNA (ScRNA), and the Ffh, and FtsY proteins. Here, we report that pneumococcal ∆scRNA, ∆ffh, and ∆ftsY mutants were significantly impaired in competence induction, competence pili production, exogenous DNA uptake, and genetic transformation. Also, the ∆scRNA mutant was significantly attenuated in the mouse models of bacteremia and pneumonia. Interestingly, unlike the ∆scRNA, both ∆ffh and ∆ftsY mutants had growth defects on Todd-Hewitt Agar, which were alleviated by the provision of free amino acids or serum. Differences in nutritional requirements between ∆ffh and ∆ftsY vs ∆scRNA suggest that Ffh and FtsY may be partially functional in the absence of ScRNA. Finally, the insertase YidC2, which could functionally rescue some SRP mutations in other streptococcal species, was not essential for pneumococcal genetic transformation. Collectively, these results indicate that ScRNA is crucial for the successful development of genetic competence and virulence in pneumococcus. IMPORTANCE: Streptococcus pneumoniae (pneumococcus) causes multiple important infectious diseases in humans. The signal recognition particle (SRP) complex, which comprised 4.5S small cytoplasmic RNA (ScRNA), and the Ffh and FtsY proteins, mediates membrane targeting and translocation of secreted proteins in all organisms. However, the role of SRP and ScRNA has not been characterized during the induction of the competence system for genetic transformation and virulence in pneumococcus. By using a combination of genetic, biochemical, proteomic, and imaging approaches, we demonstrated that the SRP complex plays a significant role in membrane targeting of competence system-regulated effectors important for genetic transformation, virulence during bacteremia and pneumonia infections, and nutritional acquisition.

Cardiac competence of the paraxial head mesoderm fades concomitant with a shift towards the head skeletal muscle programme.

The vertebrate head mesoderm provides the heart, the great vessels, some smooth and most head skeletal muscle, in addition to parts of the skull. It has been speculated that the ability to generate cardiac and smooth muscle is the evolutionary ground-state of the tissue. However, whether indeed the entire head mesoderm has generic cardiac competence, how long this may last, and what happens as cardiac competence fades, is not clear. Bone morphogenetic proteins (Bmps) are known to promote cardiogenesis. Using 41 different marker genes in the chicken embryo, we show that the paraxial head mesoderm that normally does not engage in cardiogenesis has the ability to respond to Bmp for a long time. However, Bmp signals are interpreted differently at different time points. Up to early head fold stages, the paraxial head mesoderm is able to read Bmps as signal to engage in the cardiac programme; the ability to upregulate smooth muscle markers is retained slightly longer. Notably, as cardiac competence fades, Bmp promotes the head skeletal muscle programme instead. The switch from cardiac to skeletal muscle competence is Wnt-independent as Wnt caudalises the head mesoderm and also suppresses Msc-inducing Bmp provided by the prechordal plate, thus suppressing both the cardiac and the head skeletal muscle programmes. Our study for the first time suggests a specific transition state in the embryo when cardiac competence is replaced by skeletal muscle competence. It sets the stage to unravel the cardiac-skeletal muscle antagonism that is known to partially collapse in heart failure.

Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide.

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S. mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S. mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S. mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S. mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S. mitis CSP signal can modulate QS response in S. pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S. mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S. mitis as it inhabits its natural niche.

cAMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence of Haemophilus influenzae.

Most naturally competent bacteria tightly regulate the window of the competent state to maximize their ecological fitness under specific conditions. Development of competence by Haemophilus influenzae strain Rd KW20 is stimulated by cAMP and inhibited by purine nucleotides, respectively. In contrast, cAMP inhibits cell growth, but nucleotides are important for KW20 growth. However, the mechanisms underlying the abovementioned reciprocal effects are unclear. Here, we first identified a periplasmic acid phosphatase AphAEc of Escherichia coli as a new cAMP-binding protein. We show cAMP competitively inhibits the phosphatase activities of AphAEc and its homolog protein AphAHi in the KW20 strain. Furthermore, we found cAMP inhibits two other periplasmic nonspecific phosphatases, NadNHi (which provides the essential growth factor V, NAD) and HelHi (eP4, which converts NADP to NAD) in KW20. We demonstrate cAMP inhibits cell growth rate, especially via NadNHi. On the other hand, the inhibitory effect of purine nucleotide AMP on competence was abolished in the triple deletion mutant ΔhelHiΔnadNHiΔaphAHi, but not in the single, double deletion or complemented strains. Adenosine, however, still inhibited the competence of the triple deletion mutant, demonstrating the crucial role of the three phosphatases in converting nucleotides to nucleosides and thus inhibiting KW20 competence. Finally, cAMP restored the competence inhibited by GMP in a dose-dependent manner, but not competence inhibited by guanosine. Altogether, we uncovered these three periplasmic phosphatases as the key players underlying the antagonistic effects of cAMP and purine nucleotides on both cell growth and competence development of H. influenzae.

Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control.

RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNA-protein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of ~ 88% of the transcripts and ~ 62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.