Expression patterns of housekeeping genes and tissue-specific genes in black goats across multiple tissues.

Black goats are a significant meat breed in southern China. To investigate the expression patterns and biological functions of genes in various tissues of black goats, we analyzed housekeeping genes (HKGs), tissue-specific genes (TSGs), and hub genes (HUBGs) across 23 tissues. Additionally, we analyzed HKGs in 13 tissues under different feeding conditions. We identified 2968 HKGs, including six important ones. Interestingly, HKGs in grazing black goats demonstrated higher and more stable expression levels. We discovered a total of 9912 TSGs, including 134 newly identified ones. The number of TSGs for mRNA and lncRNA were nearly equal, with 127 mRNA TSGs expressed solely in one tissue. Additionally, the predicted functions of tissue-specific long non-coding RNAs (lncRNAs) targeting mRNAs corresponded with the physiological functions of the tissues.Weighted gene co-expression network analysis (WGCNA) constructed 30 modules, where the dark grey module consists almost entirely of HKGs, and TSGs are located in modules most correlated with their respective tissues. Additionally, we identified 289 HUBGs, which are involved in regulating the physiological functions of their respective tissues. Overall, these identified HKGs, TSGs, and HUBGs provide a foundation for studying the molecular mechanisms affecting the genetic and biological processes of complex traits in black goats.

Implementation of the Methyl-Seq platform to identify tissue- and sex-specific DNA methylation differences in the rat epigenome.

Epigenomic annotations for the rat lag far behind those of human and mouse, despite the rat's immense utility in pharmacological and behavioral studies and the need to understand their epigenetic mechanisms. We have designed a targeted-enrichment method followed by next-generation sequencing (Methyl-Seq) to identify DNA methylation (DNAm) signatures across the rat genome. The design reflected an attempt to create a more comprehensive investigation of the rat epigenome, as it included promoters, CpG islands, and island shores of all RefSeq genes. In this study, we implemented the rat Methyl-Seq platform and tested its ability to distinguish differentially methylated regions (DMRs) among three different tissue types, three distinct brain regions, and, in the hippocampus, between males and females. These comparisons yielded DNAm differences of differing magnitudes, many of which were independently validated by bisulfite pyrosequencing, including autosomal regions that were predicted to show the least degree of difference in DNAm between males and females. Quantitative reverse transcription PCR revealed that most genes associated with the DMRs showed tissue-, brain region-, and sex-specific differences in expression. In particular, we found evidence for sex-specific DNAm and expression differences at Tubb6, Lrrn2, Tex26, and Sox5l1, all of which play important roles in neurodevelopment and have been implicated in studies examining sex differences. Our results demonstrate the utility of the rat Methyl-Seq platform and suggest the presence of DNAm differences between the male and female hippocampus. The rat Methyl-Seq has the potential to provide epigenomic insights into pharmacological and behavioral studies performed in the rat.

Benchmarking transcriptome deconvolution methods for estimating tissue- and cell-type-specific extracellular vesicle abundances.

Extracellular vesicles (EVs) contain cell-derived lipids, proteins and RNAs; however, determining the tissue- and cell-type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell-type-specific EV abundances can be estimated by matching the EV's transcriptome to a tissue's/cell type's expression signature using deconvolutional methods, a comparative assessment of deconvolution methods' performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from four cell lines and their EVs, in silico mixtures, 118 human plasma and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure and in silico mixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The three methods were also concordant in their tissue- and cell-type-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlighted the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell-type-specific EV abundances in biological fluids.

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the DM-domain transcription factor DMD-3. To find genes regulated by DMD-3, we performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males versus dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the unfolded protein response pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are co-regulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

Tissue-specific accumulation of PIP aquaporins of a particular heteromeric composition is part of the maize response to mycorrhiza and drought.

The systemic coordination of accumulation of plasma membrane aquaporins (PIP) was investigated in this study in relation to mycorrhized maize response to a rapid development of severe drought followed by rewatering. In non-mycorrhizal roots, drought led to a drop in PIP abundance, followed by a transient increase under rewatering, whereas leaves showed an opposite pattern. In contrast, mycorrhiza contributed to maintenance of high and stable levels of PIPs in both plant organs after an initial increase, prolonged over the irrigation period. Isoelectric focusing electrophoresis resolved up to 13 aquaporin complexes with highly reproducible pl positions across leaf and root samples, symbiotic and non-symbiotic, stressed or not. Mass spectrometry recognized in leaves and roots a different ratio of PIP1 and PIP2 subunits within 2D spots that accumulated the most. Regardless of symbiotic status, drought regulation of aquaporins in roots was manifested as the prevalence of complexes that comprise almost exclusively PIP2 monomers. In contrast, the leaf response involved enrichment in PIP1s. PIP1s are thought to enhance water transport, facilitate CO2 diffusion but also affect stomatal movements. These features, together with elevated aquaporin levels, might explain a stress tolerance mechanism observed in mycorrhizal plants, resulting in faster recovery of stomatal water conductance and CO2 assimilation rate after drought.

Nearby and non-nested genes in the human genome have more similar genotype tissue expression.

Neighboring genes within a shared promoter arrangement (i.e. opposite direction with the neighboring ends as the transcriptional start sites) are expected to have a high similarity in genotype tissue expression due to the potential overlap in the promoter region. This raises the question of whether similarity in expression profiles depends on orientation of the neighboring genes and whether there exist thresholds of locality where the similarity diminishes. Thus, in this work, we compared genotype tissue expression profiles at different genomic orientations and localities. Interestingly, there exist gene pairs in the human genome with very high or low expression similarity. Shorter chromosomes tend to have more similarly expressed genes. Also, a cluster of 3 adjacent genes within the average range of 20 to 60 kilobase pairs can have very similar expression profiles regardless of their orientations. However, when genes are nested and in opposite orientations, a lower than expected similarity was observed. Lastly, in cases where genotype tissue expression data does not exist or have low read counts (e.g. non-coding RNA), our identified influencing range can be a first estimate of the genotype tissue expression.

The epigenomic matrix of tissue-specific immune memory.

Tissue-resident memory CD8+ T cells serve as a first-line defense against many pathogens. In this issue of Immunity, Buquicchio et al. unveil the epigenomic landscapes of virus-specific CD8+ T cell subsets, highlighting common and organ-specific regulators driving their differentiation.

Fly Fam161 is an essential centriole and cilium transition zone protein with unique and diverse cell type-specific localizations.

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

BeetleAtlas: An Ontogenetic and Tissue-specific Transcriptomic Atlas of the Red Flour Beetle Tribolium castaneum.

The red flour beetle Tribolium castaneum has emerged as a powerful model in insect functional genomics. However, a major limitation in the field is the lack of a detailed spatio-temporal view of the genetic signatures underpinning the function of distinct tissues and life stages. Here, we present an ontogenetic and tissue-specific web-based resource for Tribolium transcriptomics: BeetleAtlas (https://www.beetleatlas.org). This web application provides access to a database populated with quantitative expression data for nine adult and seven larval tissues, as well as for four embryonic stages of Tribolium. BeetleAtlas allows one to search for individual Tribolium genes to obtain values of both total gene expression and enrichment in different tissues, together with data for individual isoforms. To facilitate cross-species studies, one can also use Drosophila melanogaster gene identifiers to search for related Tribolium genes. For retrieved genes there are options to identify and display the tissue expression of related Tribolium genes or homologous Drosophila genes. Five additional search modes are available to find genes conforming to any of the following criteria: exhibiting high expression in a particular tissue; showing significant differences in expression between larva and adult; having a peak of expression at a specific stage of embryonic development; belonging to a particular functional category; and displaying a pattern of tissue expression similar to that of a query gene. We illustrate how the different feaures of BeetleAtlas can be used to illuminate our understanding of the genetic mechanisms underpinning the biology of what is the largest animal group on earth.

Inflammation and Fibrosis in Progeria: Organ-Specific Responses in an HGPS Mouse Model.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder that causes accelerated aging, due to a pathogenic variant in the LMNA gene. This pathogenic results in the production of progerin, a defective protein that disrupts the nuclear lamina's structure. In our study, we conducted a histopathological analysis of various organs in the LmnaG609G/G609G mouse model, which is commonly used to study HGPS. The objective of this study was to show that progerin accumulation drives systemic but organ-specific tissue damage and accelerated aging phenotypes. Our findings show significant fibrosis, inflammation, and dysfunction in multiple organ systems, including the skin, cardiovascular system, muscles, lungs, liver, kidneys, spleen, thymus, and heart. Specifically, we observed severe vascular fibrosis, reduced muscle regeneration, lung tissue remodeling, depletion of fat in the liver, and disruptions in immune structures. These results underscore the systemic nature of the disease and suggest that chronic inflammation and fibrosis play crucial roles in the accelerated aging seen in HGPS. Additionally, our study highlights that each organ responds differently to the toxic effects of progerin, indicating that there are distinct mechanisms of tissue-specific damage.

Genetic switch selectively kills hepatocellular carcinoma cell based on microRNA and tissue-specific promoter.

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

DNA methylation correlates of chronological age in diverse human tissue types.

BACKGROUND: While the association of chronological age with DNA methylation (DNAm) in whole blood has been extensively studied, the tissue-specificity of age-related DNAm changes remains an active area of research. Studies investigating the association of age with DNAm in tissues such as brain, skin, immune cells, fat, and liver have identified tissue-specific and non-specific effects, thus, motivating additional studies of diverse human tissue and cell types. RESULTS: Here, we performed an epigenome-wide association study, leveraging DNAm data (Illumina EPIC array) from 961 tissue samples representing 9 tissue types (breast, lung, colon, ovary, prostate, skeletal muscle, testis, whole blood, and kidney) from the Genotype-Tissue Expression (GTEx) project. We identified age-associated CpG sites (false discovery rate < 0.05) in 8 tissues (all except skeletal muscle, n = 47). This included 162,002 unique hypermethylated and 90,626 hypomethylated CpG sites across all tissue types, with 130,137 (80%) hypermethylated CpGs and 74,703 (82%) hypomethylated CpG sites observed in a single tissue type. While the majority of age-associated CpG sites appeared tissue-specific, the patterns of enrichment among genomic features, such as chromatin states and CpG islands, were similar across most tissues, suggesting common mechanisms underlying cellular aging. Consistent with previous findings, we observed that hypermethylated CpG sites are enriched in regions with repressed polycomb signatures and CpG islands, while hypomethylated CpG sites preferentially occurred in non-CpG islands and enhancers. To gain insights into the functional effects of age-related DNAm changes, we assessed the correlation between DNAm and local gene expression changes to identify age-related expression quantitative trait methylation (age-eQTMs). We identified several age-eQTMs present in multiple tissue-types, including in the CDKN2A, HENMT1, and VCWE regions. CONCLUSION: Overall, our findings will aid future efforts to develop biomarkers of aging and understand mechanisms of aging in diverse human tissue types.

Extracellular vesicles carry transcriptional 'dark matter' revealing tissue-specific information.

From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid-biopsy markers. Yet, the complete transcriptomic landscape of EV-associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20-50 nt), and uncharacterized genome annotations-often denoted as the 'dark matter' of the genome. In this study, we investigate the EV-associated small unannotated RNAs that arise from endogenous genes and are part of the genomic 'dark matter', which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre- & post-prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data-based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV-RNA expression patterns emerging from the un-annotated genomic regions (UGRs) of the transcriptomes associated with tissue-specific phenotypes. We have named these novel EV-associated small RNAs as 'EV-UGRs' or "EV-dark matter". Here, we demonstrate that EV-UGR gene expressions are downregulated by ∼100 fold (FDR < 0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV-UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow-up patients. Finally, we developed a stem-loop RT-qPCR assay that validated prostate cancer-specific EV-UGRs for selective fluid-based diagnostics. Overall, using an unsupervised data driven approach, we investigate the 'dark matter' of EV-transcriptome and demonstrate that EV-UGRs carry tissue-specific Information that significantly alters pre- and post-prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV-RNAs hold promise in liquid-biopsy by avoiding highly invasive biopsy procedures in prostate cancer.

Immune surveillance of cytomegalovirus in tissues.

Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs.

TransTEx: novel tissue-specificity scoring method for grouping human transcriptome into different expression groups.

MOTIVATION: Although human tissues carry out common molecular processes, gene expression patterns can distinguish different tissues. Traditional informatics methods, primarily at the gene level, overlook the complexity of alternative transcript variants and protein isoforms produced by most genes, changes in which are linked to disease prognosis and drug resistance. RESULTS: We developed TransTEx (Transcript-level Tissue Expression), a novel tissue-specificity scoring method, for grouping transcripts into four expression groups. TransTEx applies sequential cut-offs to tissue-wise transcript probability estimates, subsampling-based P-values and fold-change estimates. Application of TransTEx on GTEx mRNA-seq data divided 199 166 human transcripts into different groups as 17 999 tissue-specific (TSp), 7436 tissue-enhanced, 36 783 widely expressed (Wide), 79 191 lowly expressed (Low), and 57 757 no expression (Null) transcripts. Testis has the most (13 466) TSp isoforms followed by liver (890), brain (701), pituitary (435), and muscle (420). We found that the tissue specificity of alternative transcripts of a gene is predominantly influenced by alternate promoter usage. By overlapping brain-specific transcripts with the cell-type gene-markers in scBrainMap database, we found that 63% of the brain-specific transcripts were enriched in nonneuronal cell types, predominantly astrocytes followed by endothelial cells and oligodendrocytes. In addition, we found 61 brain cell-type marker genes encoding a total of 176 alternative transcripts as brain-specific and 22 alternative transcripts as testis-specific, highlighting the complex TSp and cell-type specific gene regulation and expression at isoform-level. TransTEx can be adopted to the analysis of bulk RNA-seq or scRNA-seq datasets to find tissue- and/or cell-type specific isoform-level gene markers. AVAILABILITY AND IMPLEMENTATION: TransTEx database: https://bmi.cewit.stonybrook.edu/transtexdb/ and the R package is available via GitHub: https://github.com/pallavisurana1/TransTEx.

From screens to seas: Tech contaminants in tiger sharks.

The potential negative impacts of Technology-Critical Elements (TCEs) on the environment and wildlife, despite increasingly recognized, remain largely overlooked. In this sense, this study aimed to investigate the concentrations of several TCEs, including rubidium (Rb), titanium (Ti) and various Rare Earth Elements (REEs), in different tissues of tiger sharks. Sharks incidentally caught by artisanal fleets in southern Brazil were opportunistically sampled and liver, gills, kidneys, heart, muscle, eyes, brain, skin, and teeth were analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Significant Rb concentration variations were observed across different tissues, with higher levels detected in kidneys and lower levels in the liver. Titanium concentrations also exhibited significant differences, with higher levels detected in teeth and lower levels in liver. Although no statistical differences were observed for the analyzed REEs, a trend of higher accumulation in the liver, gills, and skin was noted. Light Rare Earth Elements (LREEs) were found predominantly in all organs, with neodymium, lanthanum, and cerium as the most significant REEs detected. Several statistically significant correlations were identified between Rb and REEs, as well as between Ti and REEs, indicating systemic transport of these elements across different tissues. These findings indicate that the growing extraction and disposal of metallic elements, driven by technological advancements, may lead to their assimilation by marine fauna, particularly at higher trophic levels. The potential harmful effects on these organisms remain unknown and require urgent investigation. Additionally, as mining activities intensify globally, precise legislative measures are essential to address environmental concerns, species conservation, and human health considerations.

Comprehensive analysis of single-nucleotide variants and alternative polyadenylation between inbred and outbred pigs.

Inbreeding can lead to the accumulation of homozygous single nucleotide polymorphisms (SNPs) in the genome, which can significantly affect gene expression and phenotype. In this study, we examined the impact of homozygous SNPs resulting from inbreeding on alternative polyadenylation (APA) site selection and the underlying genetic mechanisms using inbred Luchuan pigs. Genome resequencing revealed that inbreeding results in a high accumulation of homozygous SNPs within the pig genome. 3' mRNA-seq on leg muscle, submandibular lymph node, and liver tissues was performed to identify differences in APA events between inbred and outbred Luchuan pigs. We revealed different tissue-specific APA usage caused by inbreeding, which were associated with different biological processes. Furthermore, we explored the role of polyadenylation signal (PAS) SNPs in APA regulation under inbreeding and identified key genes such as PUM1, SCARF1, RIPOR2, C1D, and LRRK2 that are involved in biological processes regulation. This study provides resources and sheds light on the impact of genomic homozygosity on APA regulation, offering insights into genetic characteristics and biological processes associated with inbreeding.

Pervasive tissue-, genetic background-, and allele-specific gene expression effects in Drosophila melanogaster.

The pervasiveness of gene expression variation and its contribution to phenotypic variation and evolution is well known. This gene expression variation is context dependent, with differences in regulatory architecture often associated with intrinsic and environmental factors, and is modulated by regulatory elements that can act in cis (linked) or in trans (unlinked) relative to the genes they affect. So far, little is known about how this genetic variation affects the evolution of regulatory architecture among closely related tissues during population divergence. To address this question, we analyzed gene expression in the midgut, hindgut, and Malpighian tubule as well as microbiome composition in the two gut tissues in four Drosophila melanogaster strains and their F1 hybrids from two divergent populations: one from the derived, European range and one from the ancestral, African range. In both the transcriptome and microbiome data, we detected extensive tissue- and genetic background-specific effects, including effects of genetic background on overall tissue specificity. Tissue-specific effects were typically stronger than genetic background-specific effects, although the two gut tissues were not more similar to each other than to the Malpighian tubules. An examination of allele specific expression revealed that, while both cis and trans effects were more tissue-specific in genes expressed differentially between populations than genes with conserved expression, trans effects were more tissue-specific than cis effects. Despite there being highly variable regulatory architecture, this observation was robust across tissues and genetic backgrounds, suggesting that the expression of trans variation can be spatially fine-tuned as well as or better than cis variation during population divergence and yielding new insights into cis and trans regulatory evolution.

Development and tissue specific expression of RAPGEF1 (C3G) transcripts having exons encoding disordered segments with predicted regulatory function.

BACKGROUND: The ubiquitously expressed Guanine nucleotide exchange factor, RAPGEF1 (C3G), is essential for early development of mouse embryos. It functions to regulate gene expression and cytoskeletal reorganization, thereby controlling cell proliferation and differentiation. While multiple transcripts have been predicted, their expression in mouse tissues has not been investigated in detail. METHODS & RESULTS: Full length RAPGEF1 isoforms primarily arise due to splicing at two hotspots, one involving exon-3, and the other involving exons 12-14 incorporating amino acids immediately following the Crk binding region of the protein. These isoforms vary in expression across embryonic and adult organs. We detected the presence of unannotated, and unpredicted transcripts with incorporation of cassette exons in various combinations, specifically in the heart, brain, testis and skeletal muscle. Isoform switching was detected as myocytes in culture and mouse embryonic stem cells were differentiated to form myotubes, and embryoid bodies respectively. The cassette exons encode a serine-rich polypeptide chain, which is intrinsically disordered, and undergoes phosphorylation. In silico structural analysis using AlphaFold indicated that the presence of cassette exons alters intra-molecular interactions, important for regulating catalytic activity. LZerD based docking studies predicted that the isoforms with one or more cassette exons differ in interaction with their target GTPase, RAP1A. CONCLUSIONS: Our results demonstrate the expression of novel RAPGEF1 isoforms, and predict cassette exon inclusion as an additional means of regulating RAPGEF1 activity in various tissues and during differentiation.