Alternative Transcription Start Research Articles

Characterization and expression of the microtubule associated protein tau gene isoforms and their impact on clinical outcomes in glioma patients

Gliomas are the most common tumors found in the central nervous system. They are classified based on their histopathological malignancy. Low-grade gliomas (LGG) can turn into glioblastomas (GBM), the most aggressive form of brain cancer with the worst prognosis. Previous studies have shown that microtubule-associated protein tau (MAPT) is increased in GBM and that reducing it can improve survival in animal models. On the other hand, elevated MAPT expression has been linked to increased survival in LGG, indicating its potential as a survival biomarker. However, previous studies have primarily focused on the canonical protein-coding transcripts and gene-level expression of MAPT, disregarding the diversity of its many isoforms. Many diseases in almost every tissue have been associated with a dysregulation of alternative splicing, either through an altered ratio of the expressed isoforms or by expressing aberrant transcripts with different alternative transcription start and termination sites. This study investigates the association of specific MAPT transcripts with survival in LGG and GBM using publicly available genomic datasets and performing statistical analyses. The results of the study indicated coding and several non-coding isoforms associated with overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). After including the relevant predictors as covariates, it was observed that among the protein-coding isoforms, MAPT-205 and MAPT-201 were significantly associated with clinical survival endpoints, but with opposite trends. These findings highlight the importance of studying the role of MAPT isoforms in glioma cellular phenotypes, aggressiveness, and patient survival.

Alternative Transcripts Diversify Genome Function for Phenome Relevance to Health and Diseases.

Manipulation using alternative exon splicing (AES), alternative transcription start (ATS), and alternative polyadenylation (APA) sites are key to transcript diversity underlying health and disease. All three are pervasive in organisms, present in at least 50% of human protein-coding genes. In fact, ATS and APA site use has the highest impact on protein identity, with their ability to alter which first and last exons are utilized as well as impacting stability and translation efficiency. These RNA variants have been shown to be highly specific, both in tissue type and stage, with demonstrated importance to cell proliferation, differentiation and the transition from fetal to adult cells. While alternative exon splicing has a limited effect on protein identity, its ubiquity highlights the importance of these minor alterations, which can alter other features such as localization. The three processes are also highly interwoven, with overlapping, complementary, and competing factors, RNA polymerase II and its CTD (C-terminal domain) chief among them. Their role in development means dysregulation leads to a wide variety of disorders and cancers, with some forms of disease disproportionately affected by specific mechanisms (AES, ATS, or APA). Challenges associated with the genome-wide profiling of RNA variants and their potential solutions are also discussed in this review.

Dissecting the Isoform-Specific Roles of FTZ-F1 in the Larval-Larval and Larval-Pupal Ecdyses in Henosepilachna vigintioctopunctata.

Simple SummaryFushi Tarazu Factor-1 (FTZ-F1) plays a crucial regulatory role in molting in insects. It is hypothesized that, by alternative transcription start and splicing, the FTZ-F1 gene generates two isomers (α- and βFTZ-F1) that exert isoform-specific roles in non-Drosophilid insects. In the present paper, we first unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. We then found that αFTZ-F1 and βFTZ-F1 were actively transcribed throughout the development, from embryo to adult, in Henosepilachna vigintioctopunctata. Moreover, by RNA interference, we confirmed that both FTZ-F1 isoforms act as regulators in larval–larval molting and βFTZ-F1 is involved in the regulation of the larval–pupal transition.Fushi Tarazu Factor 1 (FTZ-F1), a member of the nuclear receptor superfamily, is the downstream factor of 20-hydroxyecdysone signaling. In Drosophila melanogaster, alternative transcription start and splicing in the FTZ-F1 gene generate αFTZ-F1 and βFTZ-F1 isoforms, which are vital for pair-rule segmentation in early embryogenesis and post-embryonic development, respectively. However, whether the same mRNA isoforms are present and exert the conservative roles remains to be clarified in other insects. In the present paper, we first mined the genomic data of representative insect species and unveiled that the same post-transcriptional processing in FTZ-F1 occurred in coleopterans, lepidopterans, dipterans and hymenopterans. Our expression data in Henosepilachna vigintioctopunctata, a serious polyphagous defoliator damaging a wide range of crops in Solanaceae and Cucurbitaceae, showed that both αFTZ-F1 and βFTZ-F1 were actively transcribed throughout the development, from embryo to adult. The RNA interference-aided knockdown of both isoforms completely arrested larval ecdysis from the third to the fourth instar, in contrast to the depletion of either isoform. In contrast, silencing βFTZ-F1, rather than αFTZ-F1, severely impaired the larval–pupal transformation. We accordingly propose that both FTZ-F1 isoforms are essential but mutually interchangeable for larval–larval molting, while βFTZ-F1 is necessary for the larval–pupal transition and sufficient to exert the role of both FTZ-F1s during larval–pupal metamorphosis in H. vigintioctopunctata.

Genome annotation with long RNA reads reveals new patterns of gene expression and improves single-cell analyses in an ant brain

BackgroundFunctional genomic analyses rely on high-quality genome assemblies and annotations. Highly contiguous genome assemblies have become available for a variety of species, but accurate and complete annotation of gene models, inclusive of alternative splice isoforms and transcription start and termination sites, remains difficult with traditional approaches.ResultsHere, we utilized full-length isoform sequencing (Iso-Seq), a long-read RNA sequencing technology, to obtain a comprehensive annotation of the transcriptome of the ant Harpegnathos saltator. The improved genome annotations include additional splice isoforms and extended 3′ untranslated regions for more than 4000 genes. Reanalysis of RNA-seq experiments using these annotations revealed several genes with caste-specific differential expression and tissue- or caste-specific splicing patterns that were missed in previous analyses. The extended 3′ untranslated regions afforded great improvements in the analysis of existing single-cell RNA-seq data, resulting in the recovery of the transcriptomes of 18% more cells. The deeper single-cell transcriptomes obtained with these new annotations allowed us to identify additional markers for several cell types in the ant brain, as well as genes differentially expressed across castes in specific cell types.ConclusionsOur results demonstrate that Iso-Seq is an efficient and effective approach to improve genome annotations and maximize the amount of information that can be obtained from existing and future genomic datasets in Harpegnathos and other organisms.

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish.

Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although short-read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full-length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq-predicted transcription start sites were more accurate and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

The FTZ-F1 gene encodes two functionally distinct nuclear receptor isoforms in the ectoparasitic copepod salmon louse (Lepeophtheirus salmonis).

The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic crustacean that annually inflicts substantial losses to the aquaculture industry in the northern hemisphere and poses a threat to the wild populations of salmonids. The salmon louse life cycle consists of eight developmental stages each separated by a molt. Fushi Tarazu Factor-1 (FTZ-F1) is an ecdysteroid-regulated gene that encodes a member of the NR5A family of nuclear receptors that is shown to play a crucial regulatory role in molting in insects and nematodes. Characterization of an FTZ-F1 orthologue in the salmon louse gave two isoforms named αFTZ-F1 and βFTZ-F1, which are identical except for the presence of a unique N-terminal domain (A/B domain). A comparison suggest conservation of the FTZ-F1 gene structure among ecdysozoans, with the exception of nematodes, to produce isoforms with unique N-terminal domains through alternative transcription start and splicing. The two isoforms of the salmon louse FTZ-F1 were expressed in different amounts in the same tissues and showed a distinct cyclical expression pattern through the molting cycle with βFTZ-F1 being the highest expressed isoform. While RNA interference knockdown of βFTZ-F1 in nauplius larvae and in pre-adult males lead to molting arrest, knockdown of βFTZ-F1 in pre-adult II female lice caused disruption of oocyte maturation at the vitellogenic stage. No apparent phenotype could be observed in αFTZ-F1 knockdown larvae, or in their development to adults, and no genes were found to be differentially expressed in the nauplii larvae following αFTZ-F1 knockdown. βFTZ-F1 knockdown in nauplii larvae caused both down and upregulation of genes associated with proteolysis and chitin binding and affected a large number of genes which are in normal salmon louse development expressed in a cyclical pattern. This is the first description of FTZ-F1 gene function in copepod crustaceans and provides a foundation to expand the understanding of the molecular mechanisms of molting in the salmon louse and other copepods.

Genome-to-phenome research in rats: progress and perspectives.

Because of their relatively short lifespan (<4 years), rats have become the second most used model organism to study health and diseases in humans who may live for up to 120 years. First-, second- and third-generation sequencing technologies and platforms have produced increasingly greater sequencing depth and accurate reads, leading to significant advancements in the rat genome assembly during the last 20 years. In fact, whole genome sequencing (WGS) of 47 strains have been completed. This has led to the discovery of genome variants in rats, which have been widely used to detect quantitative trait loci underlying complex phenotypes based on gene, haplotype, and sweep association analyses. DNA variants can also reveal strain, chromosome and gene functional evolutions. In parallel, phenome programs have advanced significantly in rats during the last 15 years and more than 10 databases host genome and/or phenome information. In order to discover the bridges between genome and phenome, systems genetics and integrative genomics approaches have been developed. On the other hand, multiple level information transfers from genome to phenome are executed by differential usage of alternative transcriptional start (ATS) and polyadenylation (APA) sites per gene. We used our own experiments to demonstrate how alternative transcriptome analysis can lead to enrichment of phenome-related causal pathways in rats. Development of advanced genome-to-phenome assays will certainly enhance rats as models for human biomedical research.

MountainClimber Identifies Alternative Transcription Start and Polyadenylation Sites in RNA-Seq.

Alternative transcription start (ATS) and alternative polyadenylation (APA) create alternative RNA isoforms and modulate many aspects of RNA expression and protein production. However, ATS and APA remain difficult to detect in RNA sequencing (RNA-seq). Here, we developed mountainClimber, a de novo cumulative-sum-based approach to identify ATS and APA as change points. Unlike many existing methods, mountainClimber runs on a single sample and identifies multiple ATS or APA sites anywhere in the transcript. We analyzed 2,342 GTEx samples (36 tissues, 215 individuals) and found that tissue type is the predominant driver of transcript end variations. 75% and 65% of genes exhibited differential APA and ATS across tissues, respectively. In particular, testis displayed longer 5' untranslated regions (UTRs) and shorter 3' UTRs, often in genes related to testis-specific biology. Overall, we report the largest study of transcript ends across human tissues to our knowledge. mountainClimber is available at github.com/gxiaolab/mountainClimber.

Abstract A72: Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia

Abstract Introduction:The RON tyrosine kinase receptor is increasingly overexpressed during progression to Pancreatic Intraepithelial Neoplasia (PanIN) and pancreatic duct adenocarcinoma (PDAC). RON expression in these neoplastic cells is associated with a highly invasive phenotype and resistance to gemcitabine. Mutations in the gene for RON, MST1R, are rare but alternative splicing produces a wide variety of protein isoforms. Exon skipping is the most common and a skipping of exon 11 produces RONΔ165. Alternative transcription start in intron 10 produces short-form RON (sfRON) which promotes metastasis in breast cancer. Eight RON isoforms have previously been reported in different epithelial cancer types (Colon, Breast, Stomach) and we initially sought to characterize their expression in PDAC. Using low passage patient derived xenografts (PDXs), we found that the known RON isoforms Δ165 and sfRON were expressed in the majority of PDX’s. Results: This study revealed a novel RON isoform resulting from partial splicing of exons 5 and 6 (P5P6) to produce a protein that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. Peptides specific to RON P5P6 were detected in pancreatic cancer specimens by mass spectrometry which confirms translation of the protein. Using end point PCR we determined that RON is overexpressed in 90% of PDX and P5P6 expressed in 73%. In PDXs absolute quantification PCR determined RON isoforms P5P6, sfRON, and RONΔ165 make up 15 – 50% of total RON transcripts. This puts total isoform expression at up to 500 times more than the full length protein in normal pancreas. To determine function of these isoforms we overexpressed sfRON, RON P5P6, and RONΔ165 in a Human Pancreatic Ductal Epithelial (HPDE) cell line using a lentiviral vector. We found that all isoforms are constitutively phosphorylated, present in the cytoplasm in a reticular pattern, and RON P5P6 traffics to the plasma membrane. RON P5P6 is spliced into α and β subunits and can bind its ligand Macrophage Stimulating Protein similar to full length RON. Expression of P5P6 in HPDE cells activates downstream AKT and MAPK signaling pathways and increases cell motility, VEGF secretion, and markers of EMT. HPDE cells expressing P5P6 and short form RON were injected orthotopically into NOD SCID Gamma mouse pancreata and are tumorigenic while the parental cell line is not. P5P6 and short form RON tumors induce a denser stroma not characteristically observed in cell line derived xenografts. In HPDE sfRON tumors, host mouse acini adjacent to the tumor undergo acinar to ductal metaplasia. As the tumor invades into the mouse pancreas these newly formed ducts become increasingly dysplastic and begin to express mesenchymal markers. Conclusion: We have identified protein isoforms of the RON tyrosine kinase receptor which are expressed in human pancreatic cancer. A novel isoform RON P5P6 and short form RON transform human pancreatic ductal epithelial cells to become tumorigenic. Expression of short form RON in these tumors causes host mouse pancreas to undergo acinar to ductal metaplasia. Ultimately these findings demonstrate that RON isoform expression by pancreatic cancer cells has significant biologic consequences and promotes interaction with the adjacent normal pancreas. Further work to characterize the soluble factors responsible for RON paracrine signaling is currently underway. Citation Format: Jeffery M. Chakedis, Randall French, Michele Babicky, Dawn Jaquish, Haleigh Howard, Evangeline Mose, Jaclyn Miyamoto, Zakk Walterscheid, Patrick Holman, Andrew M. Lowy. Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A72.

NGS-based targeted RNA sequencing for expression profiling and relative quantitation of specific gene isoforms and fusions in tumor-specific panels.

11108 Background: Gene expression signatures have become a useful tool for the identification of tumor subtypes and response to specific therapies. Expression of tumor, metastatic and macrophage specific transcripts utilizing alternative promoters and transcriptional start sites can further characterize these tumors . NGS is a powerful tool for gene expression analysis, however larger sample input requirements (&gt;100ng) and excessive sequencing depth requirements (30-40M tags/sample) to detect the expression of rare isoforms or fusions in tumor samples are prohibitive for clinical assay development. We describe the development of a targeted RNA sequencing assay for the relative quantitation of specific gene expression signatures, known splice variants and gene fusions from less than 100 ng of starting material in a single tube universal amplification format. Methods: Primers for 52 genes, isoforms and gene fusion products were designed using the universal amplification strategy. 10 ng of RNA from 5 matched tumor/adjacent normal breast cancer tumor pairs were assayed. Libraries were prepared for sequencing by emPCR and sequenced on Ion Torrent PGM. Data were aligned via TMAP. Relative expression was determined vs. housekeeping genes or wild type transcripts. Results: All gene targets were detected at significant levels in at least one tumor sample. Robust expression profiling (5 log dynamic range) was obtained from FFPE macrodissected tumor and normal samples with as little as 200K reads/sample. Immune specific transcripts demonstrated differential expression (CCL3, AIF, FCGR3A and CSF1) across patients and matched pairs as well as an upregulation of CXCL12, indicative of tumor associated macrophages. Conclusions: Targeted RNAseq demonstrates detection and quantitation of relative expression levels of not only tumor subclass specific gene expression signatures, but immune cell specific transcripts from 10ng of FFPE derived total RNA derived from macrodissected tumor samples. The lower input requirements, quicker turnaround time and incredible sensitivity of targeted RNAseq make this assay a useful tool for clinical assay development.

Oct-4B isoform is differentially expressed in breast cancer cells: hypermethylation of regulatory elements of Oct-4A suggests an alternative promoter and transcriptional start site for Oct-4B transcription

The human Oct-4 gene has three isoforms, Oct-4A, Oct-4B and Oct-4B1, which are thought to be derived from alternative splicing. It remains controversial whether the Oct-4 gene is expressed in cancer cells. Expression of Oct-4A is regulated by two elements, the PE (proximal enhancer) and DE (distal enhancer), but the expression and regulation of Oct-4B are not well known. Here, we firstly report that Oct-4B is expressed at low levels in MCF-7 cells, while the Oct-4A gene is inactivated. By analysing the function of different promoter constructs and the DNA methylation status of three regulatory regions, we demonstrate that the Oct-4A gene in MCF-7 cells is repressed by epigenetic control rather than transcriptional control. In addition, we speculate that the transcription of Oct-4B in MCF-7 cells is differentially regulated by additional regulatory elements. This work will enhance the understanding of Oct-4 gene in differential regulation.

Alternative transcripts expressed by small bristles, the Drosophila melanogaster nxf1 gene

The tissue-specific accumulation of small bristles (Dm nxf1) transcripts at different developmental stages of Drosophila melanogaster was analyzed by Northern blots and RT PCR. We identified four distinct transcripts: ubiquitous (3.5 kb); ovary and early embryo specific (3.3 kb); testis specific (1.9 kb and 2.8 kb) and nervous system specific (5.1 kb). The pattern of Dm nxf1 gene expression in ovaries and early embryos (0–2 h) is similar: the sizes of transcripts range from 3.0 to 3.5 kb. We propose that this size variability may reflect the different extent of cytoplasmic polyadenylation. In testes, the 2.8-kb transcript originates from alternative termination of transcription and the 1.9-kb transcript is supposed to originate from an alternative transcription start. During ontogenesis, the 5.1-kb transcript can be clearly detected in 10- to 18-h-old embryos, most prominently in the nervous ganglia of larvae, and it represents a major species in imago head extracts. We found that the 5.1-kb transcript, similarly to the nxf1 heavy transcripts in Homo sapiens and Mus musculus, results from the retention of intron 5–6 that corresponds to the intron 10–11 in Hs nxf1 and Mm nxf1 genes.

TRANSPARENT TESTA 12 genes from Brassica napus and parental species: cloning, evolution, and differential involvement in yellow seed trait

Molecular dissection of the Brassica yellow seed trait has been the subject of intense investigation. Arabidopsis thaliana TRANSPARENT TESTA 12 (AtTT12) encodes a multidrug and toxic compound extrusion (MATE) transporter involved in seed coat pigmentation. Two, one, and one full-length TT12 genes were isolated from B. napus, B. oleracea, and B. rapa, respectively, and Southern hybridization confirmed these gene numbers, implying loss of some of the triplicated TT12 genes in Brassica. BnTT12-1, BnTT12-2, BoTT12, and BrTT12 are 2,714, 3,062, 4,760, and 2,716 bp, with the longest mRNAs of 1,749, 1,711, 1,739, and 1,752 bp, respectively. All genes contained alternative transcriptional start and polyadenylation sites. BrTT12 and BoTT12 are the progenitors of BnTT12-1 and BnTT12-2, respectively, validating B. napus as an amphidiploid. All Brassica TT12 proteins displayed high levels of identity (>99%) to each other and to AtTT12 (>92%). Brassica TT12 genes resembled AtTT12 in such basic features as MatE/NorM CDs, subcellular localization, transmembrane helices, and phosphorylation sites. Plant TT12 orthologs differ from other MATE proteins by two specific motifs. Like AtTT12, all Brassica TT12 genes are most highly expressed in developing seeds. However, a range of organ specificity was observed with BnTT12 genes being less organ-specific. TT12 expression is absent in B. rapa yellow-seeded line 06K124, but not downregulated in B. oleracea yellow-seeded line 06K165. In B. napus yellow-seeded line L2, BnTT12-2 expression is absent, whereas BnTT12-1 is expressed normally. Among Brassica species, TT12 genes are differentially related to the yellow seed trait. The molecular basis for the yellow seed trait, in Brassica, and the theoretical and practical implications of the highly variable intron 1 of these TT12 genes are discussed.

Profiling YB-1 responsive genes in basal-like breast cancer cells by ChIP-on-chip reveals direct binding to PIK3CAs

Purpose: Basal-like breast cancers (BLBC) are typically very aggressive and they are also prone to a high rate of recurrence. We recently identified the transcription factor Y-box binding protein-1 (YB-1) as an important component of BLBC by screening breast tumour tissue microarrays. YB-1 is well known for its ability to confer chemotherapy resistance and to promote the growth of a wide range of cancer cell types. The breast cancer cell line SUM149 was characterized by us as having all the hallmarks of BLBC including a lack of the estrogen, progesterone, and Her-2 receptors. We also find that they express high levels of the epidermal growth factor receptor, a YB-1 responsive gene. &#x0D; We performed a genome-wide promoter screen of the possible YB-1 responsive genes using the NimbleGen ChIP-on-chip (COC) platform to begin to understand how this oncogene regulates the growth of basal-like breast cancer cells. YB-1 was found to bind to ~6,700 human promoters. Several members of the phosphatidylinositol-3-kinase (PI3K) pathway were identified (p110a, p85, Akt-2, ILK). Of particular interest was the identification of PIK3CA, the gene that codes for the p110a catalytic subunit of PI3K. PI3K, a lipid kinase with important roles in neoplasia, phosphorylates membrane inositol lipids resulting in activation of downstream effectors that are involved in cellular responses such as cell proliferation, survival, metabolism, and cytoskeletal reorganization. Many studies have suggested importance of the role of PI3K signaling in various types of cancer. Approximately 20% of breast cancers harbour somatic activating mutations in PIK3CA, and about 8% have amplification of PIK3CA. The remainder of the cancers have elevated levels of PI3K via other mechanisms including increased transcription. &#x0D; Methods &amp; Results: Here we propose that YB-1 overexpression in cancer leads to increased PIK3CA transcription via direct binding to the promoter. To extend our COC findings, we knocked down YB-1 with siRNA and observed a concomitant loss in p110a by immunoblotting. We have recently identified two alternate PIK3CA transcriptional start sites (exon 1a and 1b) and two alternate PIK3CA promoters (promoter 1a and 1b). We mapped potential YB-1 binding sites on PIK3CA promoters 1a and 1b, and identified five putative YB-1 binding sites within promoter 1a and one putative binding site within promoter 1b. We further performed traditional ChIP and EMSA to confirm the interaction of YB-1 with the putative binding sites on PIK3CA promoter, and determined that YB-1 binds to promoter 1a, but not to promoter 1b. &#x0D; Conclusion:The demonstration that YB-1 binds directly to the PIK3CA promoter and induces its activity identifies a novel mechanism whereby activation of YB-1 and subsequent up-regulation of PIK3CA may contribute to the pathophysiology, outcomes and therapeutic responsiveness of breast cancer.

Identification of a New Variant of PDE1A Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase Expressed in Mouse Sperm1

In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.

Gonadotropin-releasing hormone messenger RNA expression in gonadotroph tumors and normal human pituitary.

Gonadotroph tumors predominantly secrete FSH or free gonadotropin hormone subunits and rarely LH. In contrast to normal gonadotrophs, a subset of tumors synthesize FSH beta-subunit (SU) in excess of alpha-SU, and the cause of gonadotropin hormone-SU biosynthetic defects in these tumors is unknown. Gonadotropin-releasing hormone (GnRH) is known to modify gonadotropin hormone-SU biosynthesis and secretion and may be an important determinant of gonadotroph tumor hormone regulation. Data in experimental animals have demonstrated that endogenous expression of GnRH may occur in the pituitary. We therefore determined whether 1) the GnRH gene is expressed in gonadotroph tumors and normal pituitaries using reverse transcriptase (RT)-PCR; 2) the GnRH receptor gene is co-expressed in gonadotroph tumors; 3) an alternative upstream transcriptional start site on the GnRH gene is utilized; and 4) media from primary cultures of gonadotroph tumors have detectable GnRH immunoreactivity by RIA. GnRH messenger RNA (mRNA) was detected in 10/10 gonadotroph tumors, eight of which expressed GnRH-Receptor mRNA. Both mRNAs were detected in all normal pituitaries studied (n = 6). Six of 10 gonadotroph tumors and 3/6 normal pituitaries had GnRH transcripts derived from the upstream transcriptional start site (5'GnRH). GnRH immunoreactivity was detected in overnight media from 3/3 primary gonadotroph tumor cultures (range: 1.89-5.86pg/mL; limit of detection (LD) = < 1.58pg/mL) but was not detectable in control media. This is the first study to demonstrate endogenous GnRH gene expression in human pituitary adenomas and normal human pituitary tissue. The presence of both GnRH and GnRH-Rc suggest that GnRH may be a paracrine/autocrine regulator of cell function in the pituitary and may affect gonadotroph tumor hormone phenotype.

Unusual structure, evolutionary conservation of non-coding sequences and numerous pseudogenes characterize the human H3.3 histone multigene family.

The genomic organization of the replication-independent, basally expressed, human H3.3 gene is atypical of traditional histone gene organization. The gene contains 3 introns totalling 7.8 kb and unusual direct repeats flank all three intron-exon splice junctions. The transcription initiation site was mapped by S1 nuclease protection analysis and confirms that cDNA clones previously reported were full length. Sequence similarities between regions at the 5' and 3' termini of this human gene and a chicken H3.3 gene lead us to propose that either the previous assignments of termini of the chicken gene are in error, or there are alternative transcription start and polyadenylation sites. The 85% base matching of human and chicken H3.3 3'UTR sequences for 520 bases is unprecedented among homolog 3'UTR segments, especially considering that these species are separated by over 250 Myr of evolution. We also present the sequence of three related processed human H3.3 pseudogenes and provide evidence demonstrating that most of the 20 to 30 copies of the H3.3 gene within the human genome are in fact processed pseudogenes.