The proteasome regulator Rpn4 controls antifungal drug tolerance by coupling protein homeostasis with metabolic responses to drug stress.

Fungal pathogens overcome antifungal drug therapy by classic resistance mechanisms, such as increased efflux or changes to the drug target. However, even when a fungal strain is susceptible, trailing or persistent microbial growth in the presence of an antifungal drug can contribute to therapeutic failure. This trailing growth is caused by adaptive physiological changes that enable the growth of a subpopulation of fungal cells in high drug concentrations, in what is described as drug tolerance. Mechanistically, antifungal drug tolerance is incompletely understood. Here we report that the transcriptional activator Rpn4 is important for drug tolerance in the human fungal pathogen Candida albicans. Deletion of RPN4 eliminates tolerance to the commonly used antifungal drug fluconazole. We defined the mechanism and show that Rpn4 controls fluconazole tolerance via two target pathways. First, Rpn4 activates proteasome gene expression, which enables sufficient proteasome capacity to overcome fluconazole-induced proteotoxicity and the accumulation of ubiquitinated proteins targeted for degradation. Consistently, inhibition of the proteasome with MG132 eliminates fluconazole tolerance and resistance, and phenocopies the rpn4Δ/Δ mutant for loss of tolerance. Second, Rpn4 is required for wild type expression of the genes required for the synthesis of the membrane lipid ergosterol. Our data indicates that this function of Rpn4 is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. Based on our findings, we propose that Rpn4 is a central hub for fluconazole tolerance in C. albicans by coupling the regulation of protein homeostasis (proteostasis) and lipid metabolism to overcome drug-induced proteotoxicity and membrane stress.

Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.

The RSC (Remodels the Structure of Chromatin) complex of Candida albicans shows compositional divergence with distinct roles in regulating pathogenic traits.

Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. Biochemical analysis showed that CaRSC comprises 13 subunits and contains two novel non-essential members, which we named Nri1 and Nri2 (Novel RSC Interactors) that are exclusive to the CTG clade of Saccharomycotina. Genetic analysis showed distinct essentiality of C. albicans RSC subunits compared to model fungal species suggesting functional and structural divergence of RSC functions in this fungal pathogen. Transcriptomic and proteomic profiling of a conditional mutant of the essential catalytic subunit gene STH1 demonstrated global roles of RSC in C. albicans biology, with the majority of growth-related processes affected, as well as mis-regulation of genes involved in morphotype switching, host-pathogen interaction and adaptive fitness. We further assessed the functions of non-essential CaRSC subunits, showing that the novel subunit Nri1 and the bromodomain subunit Rsc4 play roles in filamentation and stress responses; and also interacted at the genetic level to regulate cell viability. Consistent with these roles, Rsc4 is required for full virulence of C. albicans in the murine model of systemic infection. Taken together, our data builds the first comprehensive study of the composition and roles of RSC in C. albicans, showing both conserved and distinct features compared to model fungal systems. The study illuminates how C. albicans uses RSC-dependent transcriptional regulation to respond to environmental signals and drive survival fitness and virulence in mammals.

Metabolic competition between host and pathogen dictates inflammasome responses to fungal infection.

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.

Mitochondrial Control of Fungal Cell Walls: Models and Relevance in Fungal Pathogens.

Proper structure and function of the fungal cell wall are controlled by metabolic processes, as well as an interplay between a range of cellular organelles. Somewhat surprisingly, mitochondrial function has been shown to be important for proper cell wall biogenesis and integrity. Mitochondria also play a role in the susceptibility of fungi to cell wall-targeting drugs. This is true in a range of fungal species, including important human fungal pathogens. The biochemical mechanisms that explain the roles of mitochondria in cell wall biology have remained elusive, but studies to date strongly support the idea that mitochondrial control over cellular lipid homeostasis is at the core of these processes. Excitingly, recent evidence suggests that the mitochondria-lipid linkages drive resistance to the echinocandin drug caspofungin, a clinically important therapeutic that targets cell wall biosynthesis. Here, we review the state of affairs in mitochondria-fungal cell wall research and propose models that could be tested in future studies. Elucidating the mechanisms that drive fungal cell wall integrity through mitochondrial functions holds promise for developing new strategies to combat fungal infections, including the possibility to potentiate the effects of antifungal drugs and curb drug resistance.

Central metabolic interactions of immune cells and microbes: prospects for defeating infections.

Antimicrobial drug resistance is threatening to take us to the "pre-antibiotic era", where people are dying from preventable and treatable diseases and the risk of hospital-associated infections compromises the success of surgery and cancer treatments. Development of new antibiotics is slow, and alternative approaches to control infections have emerged based on insights into metabolic pathways in host-microbe interactions. Central carbon metabolism of immune cells is pivotal in mounting an effective response to invading pathogens, not only to meet energy requirements, but to directly activate antimicrobial responses. Microbes are not passive players here-they remodel their metabolism to survive and grow in host environments. Sometimes, microbes might even benefit from the metabolic reprogramming of immune cells, and pathogens such as Candida albicans, Salmonella Typhimurium and Staphylococcus aureus can compete with activated host cells for sugars that are needed for essential metabolic pathways linked to inflammatory processes. Here, we discuss how metabolic interactions between innate immune cells and microbes determine their survival during infection, and ways in which metabolism could be manipulated as a therapeutic strategy.

Mdivi-1 and mitochondrial fission: recent insights from fungal pathogens.

Mitochondrial fission shows potential as a therapeutic target in non-infectious human diseases. The compound mdivi-1 was identified as a mitochondrial fission inhibitor that acts against the evolutionarily conserved mitochondrial fission GTPase Dnm1/Drp1, and shows promising data in pre-clinical models of human pathologies. Two recent studies, however, found no evidence that mdivi-1 acts as a mitochondrial fission inhibitor and proposed other mechanisms. In mammalian cells, Bordt et al. showed that mdivi-1 inhibits complex I in mitochondria (Dev Cell 40:583, 2017). In a second study, we have recently demonstrated that mdivi-1 does not trigger a mitochondrial morphology change in the human yeast pathogen Candida albicans, but impacts on endogenous nitric oxide (NO) levels and inhibits the key virulence property of hyphal formation (Koch et al., Cell Rep 25:2244, 2018). Here we discuss recent insights into mdivi-1's action in pathogenic fungi and the potential and challenges for repurposing it as an anti-infective. We also outline recent findings on the roles of mitochondrial fission in human and plant fungal pathogens, with the goal of starting the conversation on whether the research field of fungal pathogenesis can benefit from efforts in other disease areas aimed at developing therapeutic inhibitors of mitochondrial division.

Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

PAT-seq: a method to study the integration of 3'-UTR dynamics with gene expression in the eukaryotic transcriptome.

A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide.

Post-transcriptional gene regulation in the biology and virulence of Candida albicans.

In the human fungal pathogen Candida albicans, remodelling of gene expression drives host adaptation and virulence. Recent studies revealed that in addition to transcription, post-transcriptional mRNA control plays important roles in virulence-related pathways. Hyphal morphogenesis, biofilm formation, stress responses, antifungal drug susceptibility and virulence in animal models require post-transcriptional regulators. This includes RNA binding proteins that control mRNA localization, decay and translation, as well as the cytoplasmic mRNA decay pathway. Comprehensive understanding of how modulation of gene expression networks drives C. albicans virulence will necessitate integration of our knowledge on transcriptional and post-transcriptional mRNA control.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms.

OBJECTIVES: Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. METHODS: We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. RESULTS: Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide ß-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. CONCLUSIONS: Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.

The ins and outs of the intermembrane space: diverse mechanisms and evolutionary rewiring of mitochondrial protein import routes.

BACKGROUND: Mitochondrial biogenesis is an essential process in all eukaryotes. Import of proteins from the cytosol into mitochondria is a key step in organelle biogenesis. Recent evidence suggests that a given mitochondrial protein does not take the same import route in all organisms, suggesting that pathways of mitochondrial protein import can be rewired through evolution. Examples of this process so far involve proteins destined to the mitochondrial intermembrane space (IMS). SCOPE OF REVIEW: Here we review the components, substrates and energy sources of the known mechanisms of protein import into the IMS. We discuss evolutionary rewiring of the IMS import routes, focusing on the example of the lactate utilisation enzyme cytochrome b2 (Cyb2) in the model yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. MAJOR CONCLUSIONS: There are multiple import pathways used for protein entry into the IMS and they form a network capable of importing a diverse range of substrates. These pathways have been rewired, possibly in response to environmental pressures, such as those found in the niches in the human body inhabited by C. albicans. GENERAL SIGNIFICANCE: We propose that evolutionary rewiring of mitochondrial import pathways can adjust the metabolic fitness of a given species to their environmental niche. This article is part of a Special Issue entitled Frontiers of Mitochondrial.

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake.

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg(2+) levels. Populations treated with BPTI had fewer cells in S-phase of the cell cycle and a corresponding increase of cells in G(0)/G(1) and G(2) phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.

ifet-1 is a broad-scale translational repressor required for normal P granule formation in C. elegans.

Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.

System-level impact of mitochondria on fungal virulence: to metabolism and beyond.

The mitochondrion plays wide-ranging roles in eukaryotic cell physiology. In pathogenic fungi, this central metabolic organelle mediates a range of functions related to disease, from fitness of the pathogen to developmental and morphogenetic transitions to antifungal drug susceptibility. In this review, we present the latest findings in this area. We focus on likely mechanisms of mitochondrial impact on fungal virulence pathways through metabolism and stress responses, but also potentially via control over signaling pathways. We highlight fungal mitochondrial proteins that lack human homologs, and which could be inhibited as a novel approach to antifungal drug strategy.

Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins.

Fungal disease is an increasing problem in both agriculture and human health. Treatment of human fungal disease involves the use of chemical fungicides, which generally target the integrity of the fungal plasma membrane or cell wall. Chemical fungicides used for the treatment of plant disease, have more diverse mechanisms of action including inhibition of sterol biosynthesis, microtubule assembly and the mitochondrial respiratory chain. However, these treatments have limitations, including toxicity and the emergence of resistance. This has led to increased interest in the use of antimicrobial peptides for the treatment of fungal disease in both plants and humans. Antimicrobial peptides are a diverse group of molecules with differing mechanisms of action, many of which remain poorly understood. Furthermore, it is becoming increasingly apparent that stress response pathways are involved in the tolerance of fungi to both chemical fungicides and antimicrobial peptides. These signalling pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli, such as cell wall instability, changes in osmolarity and production of reactive oxygen species. Here we review stress signalling induced by treatment of fungi with chemical fungicides and antifungal peptides. Study of these pathways gives insight into how these molecules exert their antifungal effect and also into the mechanisms used by fungi to tolerate sub-lethal treatment by these molecules. Inactivation of stress response pathways represents a potential method of increasing the efficacy of antifungal molecules.

A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways.

The controlled biogenesis of mitochondria is a key cellular system coordinated with the cell division cycle, and major efforts in systems biology currently are directed toward understanding of the control points at which this coordination is achieved. Here we present insights into the function, evolution, and regulation of mitochondrial biogenesis through the study of the protein import machinery in the human fungal pathogen, Candida albicans. Features that distinguish C. albicans from baker's yeast (Saccharomyces cerevisiae) include the stringency of metabolic control at the level of oxygen consumption, the potential for ATP exchange through the porin in the outer membrane, and components and domains in the sorting and assembling machinery complex, a molecular machine that drives the assembly of proteins in the outer mitochondrial membrane. Analysis of targeting sequences and assays of mitochondrial protein import show that components of the electron transport chain are imported by distinct pathways in C. albicans and S. cerevisiae, representing an evolutionary rewiring of mitochondrial import pathways. We suggest that studies using this pathogen as a model system for mitochondrial biogenesis will greatly enhance our knowledge of how mitochondria are made and controlled through the course of the cell-division cycle.

The functions of Mediator in Candida albicans support a role in shaping species-specific gene expression.

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.

A global virulence regulator in Acinetobacter baumannii and its control of the phenylacetic acid catabolic pathway.

BACKGROUND: Acinetobacter baumannii is one of the most notorious hospital-acquired pathogens, and novel treatment strategies are desperately required. Two-component regulatory systems represent potential therapeutic targets as they mediate microorganism adaptation to changing environments, often control virulence, and are specific to bacteria. Here we describe the first global virulence regulator in A. baumannii. METHODS AND RESULTS: Using transcriptional profiling and functional assays of a deletion mutant in the A. baumannii sensor kinase gene, A1S_0574 (termed as gacS), we show that this sensor kinase regulates key virulence characteristics, including pili synthesis, biofilms, and motility, resulting in virulence attenuation in a mammalian septicemia model. Notably, we also identified that GacS regulates an operon novel to A. baumannii (paa operon), which is responsible for the metabolism of aromatic compounds. Deletion of paaE (A1S_1340) confirmed the role of this operon in A. baumannii virulence. Finally, we identified the cognate response regulator (A1S_0236) for GacS and confirmed their interaction. A1S_0236 was shown to regulate 75% of the GacS transcriptome and the same virulence phenotypes. Overexpression of A1S_0236 restored virulence in the gacS mutant. CONCLUSIONS: Our study characterizes a global virulence regulator, which may provide an alternate therapeutic target, in one of the most troublesome hospital-acquired pathogens.

ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications.

The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.