Absolute Quantification of mRNA Isoforms in Adult Stem Cells Using Microfluidic Digital PCR.

Adult stem cells play key roles in homeostasis and tissue repair. These cells are regulated by a tight control of transcriptional programs. For example, muscle stem cells (MuSCs), located beneath the basal lamina, exist in the quiescent state but can transition to an activated, proliferative state upon injury. The control of MuSC state depends on the expression levels of myogenic transcription factors. Recent studies revealed the presence of different mRNA isoforms, with distinct biological regulation. Quantifying the exact expression levels of the mRNA isoforms encoding these myogenic transcription factors is therefore key to understanding how MuSCs switch between cell states. Previously, quantitative real-time polymerase chain reaction (qRT-PCR) has been used to quantify RNA expression levels. However, qRT-PCR depends on large amounts of RNA input and only measures relative abundance. Here, we present a protocol for the absolute quantification of mRNA isoforms using microfluidic digital PCR (mdPCR). Primary MuSCs isolated from individual skeletal muscles (gastrocnemius and masseter) are lysed, and their RNA is reverse-transcribed into cDNA and copied into double-stranded DNA. Following exonuclease I digestion to remove remaining single-stranded DNA, the samples are loaded onto a mdPCR chip with TaqMan probes targeting the mRNA isoforms of interest, whereupon target molecules are amplified in nanoliter chambers. We demonstrate that mdPCR can give exact molecule counts per cell for mRNA isoforms encoding the myogenic transcription factor Pax3. This protocol enables the absolute quantification of low abundant mRNA isoforms in a fast, precise, and reliable way.

Docetaxel Resistance in Castration-Resistant Prostate Cancer: Transcriptomic Determinants and the Effect of Inhibiting Wnt/ß-Catenin Signaling by XAV939.

Castration-resistant prostate cancer (CRPC) is a common form of prostate cancer in which docetaxel-based chemotherapy is used as the first line. The present study is devoted to the analysis of transcriptome profiles of tumor cells in the development of resistance to docetaxel as well as to the assessment of the combined effect with the XAV939 tankyrase inhibitor on maintaining the sensitivity of tumor cells to chemotherapy. RNA-Seq was performed for experimental PC3 cell lines as well as for plasma exosome samples from patients with CRPC. We have identified key biological processes and identified a signature based on the expression of 17 mRNA isoforms associated with the development of docetaxel resistance in PC3 cells. Transcripts were found in exosome samples, the increased expression of which was associated with the onset of progression of CRPC during therapy. The suppression of pathways associated with the participation of cellular microtubules has also been shown when cells are treated with docetaxel in the presence of XAV939. These results highlight the importance of further research into XAV939 as a therapeutic agent in the treatment of CRPC; moreover, we have proposed a number of mRNA isoforms with high predictive potential, which can be considered as promising markers of response to docetaxel.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

Differences in the Expression Patterns of TGFß Isoforms and Associated Genes in Astrocytic Brain Tumors.

Genes associated with the TGFß isoforms are involved in a number of different cancers, and their effect on the progression of brain tumors is also being discussed. Using an oligonucleotide microarray method, we assessed differences in expression patterns of genes in astrocytic brain tumor sections from 43 patients at different stages of disease. Quantitative mRNA assessment of the three TGFß isoforms was also performed by real-time RT-qPCR. Oligonucleotide microarray data were analyzed using the PL-Grid Infrastructure. The microarray analysis showed a statistically significant (p < 0.05) increase in TGFß1 and TGFß2 expression in G3/G4 stage relative to G2, whereas real-time RT-qPCR validation confirmed this change only for the TGFß2 isoform (p < 0.05). The oligonucleotide microarray method allowed the identification of 16 differential genes associated with TGFß isoforms. Analysis of the STRING database showed that the proteins encoded by the analyzed genes form a strong interaction network (p < 0.001), and a significant number of proteins are involved in carcinogenesis. Differences in expression patterns of transcripts associated with TGFß isoforms confirm that they play a role in astrocytic brain tumor transformation. Quantitative assessment of TGFß2 mRNA may be a valuable method to complement the diagnostic process in the future.

A compensatory link between cleavage/polyadenylation and mRNA turnover regulates steady-state mRNA levels in yeast.

Cells have compensatory mechanisms to coordinate the rates of major biological processes, thereby permitting growth in a wide variety of conditions. Here, we uncover a compensatory link between cleavage/polyadenylation in the nucleus and messenger RNA (mRNA) turnover in the cytoplasm. On a global basis, same-gene 3' mRNA isoforms with twofold or greater differences in half-lives have steady-state mRNA levels that differ by significantly less than a factor of 2. In addition, increased efficiency of cleavage/polyadenylation at a specific site is associated with reduced stability of the corresponding 3' mRNA isoform. This inverse relationship between cleavage/polyadenylation and mRNA isoform half-life reduces the variability in the steady-state levels of mRNA isoforms, and it occurs in all four growth conditions tested. These observations suggest that during cleavage/polyadenylation in the nucleus, mRNA isoforms are marked in a manner that persists upon translocation to the cytoplasm and affects the activity of mRNA degradation machinery, thus influencing mRNA stability.

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. RESULTS: To identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. CONCLUSIONS: Long-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts.

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While "exitrons" are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed "falsitrons," that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.

Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells.

Transcription termination in eukaryotic cells involves the recognition of polyadenylation signals (PAS) that signal the site of pre-mRNA cleavage and polyadenylation. Most eukaryotic genes contain multiple PAS that are used by alternative polyadenylation (APA), a co-transcriptional process that increases transcriptomic diversity and modulates the fate of the mRNA and protein produced. However, current tools to pinpoint the relationship between mRNAs in different subcellular fractions and the gene expression outcome are lacking, particularly in primary human immune cells, which, due to their nature, are challenging to study. Here, we describe an integrative approach using subcellular fractionation and RNA isolation, chromatin-bound and nucleoplasmic RNA-Sequencing, 3' RNA-Sequencing and bioinformatics, to identify accurate APA mRNA isoforms and to quantify gene expression in primary human macrophages. Our protocol includes macrophage differentiation and polarization, co-culture with cancer cells, and gene silencing by siRNA. This method allows the simultaneous identification of macrophage APA mRNA isoforms integrated with the characterization of nuclear APA events, the identification of the molecular mechanisms involved, as well as the gene expression alterations caused by the cancer-macrophage crosstalk. With this methodology we identified macrophage APA mRNA signatures driven by the cancer cells that alter the macrophage inflammatory and transcriptomic profiles, with consequences for macrophage physiology and tumor evasion.

Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing nonsense-mediated decay pathways.

BACKGROUND: Alternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. RESULTS: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compare exon junction complex RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify thousands of previously unannotated splicing events; while many can be attributed to splicing noise, others are evolutionarily conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. CONCLUSIONS: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.

Proteomic and Transcriptomic Analysis Identify Spliceosome as a Significant Component of the Molecular Machinery in the Pituitary Tumors Derived from POU1F1- and NR5A1-Cell Lineages.

BACKGROUND: Pituitary adenomas (PA) are the second most common tumor in the central nervous system and have low counts of mutated genes. Splicing occurs in 95% of the coding RNA. There is scarce information about the spliceosome and mRNA-isoforms in PA, and therefore we carried out proteomic and transcriptomic analysis to identify spliceosome components and mRNA isoforms in PA. METHODS: Proteomic profile analysis was carried out by nano-HPLC and mass spectrometry with a quadrupole time-of-flight mass spectrometer. The mRNA isoforms and transcriptomic profiles were carried out by microarray technology. With proteins and mRNA information we carried out Gene Ontology and exon level analysis to identify splicing-related events. RESULTS: Approximately 2000 proteins were identified in pituitary tumors. Spliceosome proteins such as SRSF1, U2AF1 and RBM42 among others were found in PA. These results were validated at mRNA level, which showed up-regulation of spliceosome genes in PA. Spliceosome-related genes segregate and categorize PA tumor subtypes. The PA showed alterations in CDK18 and THY1 mRNA isoforms which could be tumor specific. CONCLUSIONS: Spliceosome components are significant constituents of the PA molecular machinery and could be used as molecular markers and therapeutic targets. Splicing-related genes and mRNA-isoforms profiles characterize tumor subtypes.

3'READS + RIP defines differential Staufen1 binding to alternative 3'UTR isoforms and reveals structures and sequence motifs influencing binding and polysome association.

Staufen1 (STAU1) is an RNA-binding protein (RBP) that interacts with double-stranded RNA structures and has been implicated in regulating different aspects of mRNA metabolism. Previous studies have indicated that STAU1 interacts extensively with RNA structures in coding regions (CDSs) and 3'-untranslated regions (3'UTRs). In particular, duplex structures formed within 3'UTRs by inverted-repeat Alu elements (IRAlus) interact with STAU1 through its double-stranded RNA-binding domains (dsRBDs). Using 3' region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3'READS + RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3'UTRs. Overall, relative to short 3'UTR counterparts, longer 3'UTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specific IRAlus sequences. Nevertheless, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3'UTRs that may recruit antagonistic RBPs and/or destabilize RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3'UTRs generally enhances polysome association. However, IRAlus generally have little impact on STAU1-mediated polysome association despite having strong interactions with the protein. Taken together, our work reveals complex interactions of STAU1 with its cognate RNA substrates. Our data also shed light on distinct post-transcriptional fates for the widespread APA isoforms in mammalian cells.

Computational Methods for Predicting Functions at the mRNA Isoform Level.

Multiple mRNA isoforms of the same gene are produced via alternative splicing, a biological mechanism that regulates protein diversity while maintaining genome size. Alternatively spliced mRNA isoforms of the same gene may sometimes have very similar sequence, but they can have significantly diverse effects on cellular function and regulation. The products of alternative splicing have important and diverse functional roles, such as response to environmental stress, regulation of gene expression, human heritable, and plant diseases. The mRNA isoforms of the same gene can have dramatically different functions. Despite the functional importance of mRNA isoforms, very little has been done to annotate their functions. The recent years have however seen the development of several computational methods aimed at predicting mRNA isoform level biological functions. These methods use a wide array of proteo-genomic data to develop machine learning-based mRNA isoform function prediction tools. In this review, we discuss the computational methods developed for predicting the biological function at the individual mRNA isoform level.

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data.

High-throughput single-cell RNA-seq (scRNA-seq) is a powerful tool for studying gene expression in single cells. Most current scRNA-seq bioinformatics tools focus on analysing overall expression levels, largely ignoring alternative mRNA isoform expression. We present a computational pipeline, Sierra, that readily detects differential transcript usage from data generated by commonly used polyA-captured scRNA-seq technology. We validate Sierra by comparing cardiac scRNA-seq cell types to bulk RNA-seq of matched populations, finding significant overlap in differential transcripts. Sierra detects differential transcript usage across human peripheral blood mononuclear cells and the Tabula Muris, and 3 'UTR shortening in cardiac fibroblasts. Sierra is available at https://github.com/VCCRI/Sierra .

Isoform-specific NF1 mRNA levels correlate with disease severity in Neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is characterized by an extreme clinical variability both within and between families that cannot be explained solely by the nature of the pathogenic NF1 gene mutations. A proposed model hypothesizes that variation in the levels of protein isoforms generated via alternative transcript processing acts as modifier and contributes to phenotypic variability. RESULTS: Here we used real-time quantitative PCR to investigate the levels of two major NF1 mRNA isoforms encoding proteins differing in their ability to control RAS signaling (isoforms I and II) in the peripheral blood leukocytes of 138 clinically well-characterized NF1 patients and 138 aged-matched healthy controls. As expected, expression analysis showed that NF1 isoforms I and II levels were significantly lower in patients than controls. Notably, these differences were more evident when patients were stratified according to the severity of phenotype. Moreover, a correlation was identified when comparing the levels of isoform I mRNA and the severity of NF1 features, with statistically significant lower levels associated with a severe phenotype (i.e., occurrence of learning disability/intellectual disability, optic gliomas and/or other neoplasias, and/or cerebrovascular disease) as well as in patients with cognitive impairment. CONCLUSIONS: The present findings provide preliminary evidence for a role of circuits controlling NF1 transcript processing in modulating NF1 expressivity, and document an association between the levels of neurofibromin isoform I mRNA and the severity of phenotype and cognitive impairment in NF1.

Probing In Vivo Structure of Individual mRNA 3' Isoforms Using Dimethyl Sulfate.

The DMS region extraction and deep sequencing (DREADS) procedure was designed to probe RNA structure in vivo and to link this structural information to specific 3' isoforms. Growing cells are treated with the alkylating agent dimethyl sulfate (DMS), which enters easily into cells and modifies RNA molecules at solvent-exposed A and C residues. RNA is isolated, and sequencing libraries are constructed in a manner that preserves the identities of individual mRNA isoforms arising from alternative cleavage/polyadenylation sites. During the cDNA synthesis step of library construction, the progress of reverse transcriptase (RT) is blocked when it encounters a DMS modification on the RNA, leading to disproportionate cDNA termination adjacent to DMS-modified positions. After paired-end deep sequencing, the downstream end of each sequenced fragment is mapped to a specific cleavage/poly(A) site representing an individual mRNA 3' isoform. The upstream mapped end of the sequenced fragment defines where the RT reaction stopped. Over the population of all sequenced fragments derived from a particular isoform, A and C positions that are overrepresented next to the upstream endpoints in the DMS sample (relative to a parallel untreated control) are inferred to have been DMS modified, and hence solvent exposed. This method thus allows in vivo structural information obtained using DMS to be linked to individual mRNA 3' isoforms. © 2019 by John Wiley & Sons, Inc.

Alternative polyadenylation produces multiple 3' untranslated regions of odorant receptor mRNAs in mouse olfactory sensory neurons.

BACKGROUND: Odorant receptor genes constitute the largest gene family in mammalian genomes and this family has been extensively studied in several species, but to date far less attention has been paid to the characterization of their mRNA 3' untranslated regions (3'UTRs). Given the increasing importance of UTRs in the understanding of RNA metabolism, and the growing interest in alternative polyadenylation especially in the nervous system, we aimed at identifying the alternative isoforms of odorant receptor mRNAs generated through 3'UTR variation. RESULTS: We implemented a dedicated pipeline using IsoSCM instead of Cufflinks to analyze RNA-Seq data from whole olfactory mucosa of adult mice and obtained an extensive description of the 3'UTR isoforms of odorant receptor mRNAs. To validate our bioinformatics approach, we exhaustively analyzed the 3'UTR isoforms produced from 2 pilot genes, using molecular approaches including northern blot and RNA ligation mediated polyadenylation test. Comparison between datasets further validated the pipeline and confirmed the alternative polyadenylation patterns of odorant receptors. Qualitative and quantitative analyses of the annotated 3' regions demonstrate that 1) Odorant receptor 3'UTRs are longer than previously described in the literature; 2) More than 77% of odorant receptor mRNAs are subject to alternative polyadenylation, hence generating at least 2 detectable 3'UTR isoforms; 3) Splicing events in 3'UTRs are restricted to a limited subset of odorant receptor genes; and 4) Comparison between male and female data shows no sex-specific differences in odorant receptor 3'UTR isoforms. CONCLUSIONS: We demonstrated for the first time that odorant receptor genes are extensively subject to alternative polyadenylation. This ground-breaking change to the landscape of 3'UTR isoforms of Olfr mRNAs opens new avenues for investigating their respective functions, especially during the differentiation of olfactory sensory neurons.

The Human TET2 Gene Contains Three Distinct Promoter Regions With Differing Tissue and Developmental Specificities.

Tet methylcytosine dioxygenase 2 (TET2) is a tumor suppressor gene that is inactivated in a wide range of hematological cancers. TET2 enzymatic activity converts 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC), an essential step in DNA demethylation. Human TET2 is highly expressed in pluripotent cells and down-regulated in differentiated cells: however, transcriptional regulation of the human TET2 gene has not been investigated in detail. Here we define three promoters within a 2.5 kb region located ∼ 87 kb upstream of the first TET2 coding exon. The three promoters, designated as Pro1, Pro2, and Pro3, generate three alternative first exons, and their presence in TET2 mRNAs varies with cell type and developmental stage. In general, all three TET2 transcripts are more highly expressed in human tissues rich in hematopoietic stem cells, such as spleen and bone marrow, compared to other tissues, such as brain and kidney. Transcripts from Pro2 are expressed by a broad range of tissues and at a significantly higher level than Pro1 or Pro3 transcripts. Pro3 transcripts were highly expressed by embryoid bodies generated from the H9 ES cell line, and the major Pro3 transcript is an alternatively spliced mRNA isoform that produces a truncated TET2 protein lacking the catalytic domain. Our study demonstrates distinct tissue-specific mechanisms of TET2 transcriptional regulation during early pluripotent states and in differentiated cell types.

Experiments with Snails Add to Our Knowledge about the Role of aPKC Subfamily Kinases in Learning.

Protein kinase Mζ is considered important for memory formation and maintenance in different species, including invertebrates. PKMζ participates in multiple molecular pathways in neurons, regulating translation initiation rate, AMPA receptors turnover, synaptic scaffolding assembly, and other processes. Here, for the first time, we established the sequence of mRNA encoding PKMζ homolog in land snail Helix lucorum. We annotated important features of this mRNA: domains, putative capping sites, translation starts, and splicing sites. We discovered that this mRNA has at least two isoforms, and one of them lacks sequence encoding C1 domain. C1 deletion may be unique for snail because it has not been previously found in other species. We performed behavioral experiments with snails, measured expression levels of identified isoforms, and confirmed that their expression correlates with one type of learning.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

PD-1 in human NK cells: evidence of cytoplasmic mRNA and protein expression.

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1+ NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1-, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56dim than in CD56bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.