Proinsulin is encoded by an RNA splice variant in human blood myeloid cells

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of immunological self and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

Semantic annotation support in the absence of consensus

We are interested in the annotation of knowledge which does not necessarily require a consensus. Scholarly debate is an example of such a category of knowledge where disagreement and contest are widespread and desirable, and unlike many Semantic Web approaches, we are interested in the capture and the compilation of these conflicting viewpoints and perspectives. The Scholarly Ontologies project provides the underlying formalism to represent this meta-knowledge, and we will look at ways to lighten the burden of its creation. After having described some particularities of this kind of knowledge, we introduce ClaimSpotter, our approach to support its ‘capture’, based on the elicitation of a number of recommendations which are presented for consideration to our annotators (or analysts), and give some elements of evaluation.

A Rare Loss-of-Function Variant in Scavenger Receptor Class B Type I (SCARB1) Raises HDL Cholesterol and Increases Risk of Coronary Disease

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL)
cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a
lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI
knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased
atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains
unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328
individuals with extremely high plasma HDL-C levels, we identified a homozygote for a lossof-function
variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene
encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and
abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells
derived from induced pluripotent stem cells from the homozygous subject, and in mice.
Large population-based studies revealed that subjects who are heterozygous carriers of
the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have
a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is
statistically significant).

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Investigation of acceptor dopants in ZrNiSn half-Heusler materials

Thermoelectric generators (TEG) are solid state devices and are able to convert thermal energy directly into electricity and thus could play an important role in waste heat recovery in the near future. Half-Heusler (HH) compounds with the general formula MNiSn (M = Ti, Zr, Hf) built a promising class of materials for these applications because of their high Seebeck coefficients, their environmentally friendliness and their cost advantage over conventional thermoelectric materials.rnrnMuch of the existing literature on HH deals with thermoelectric characterization of n-type MNiSn and p-type MCoSb compounds. Studies on p-type MNiSn-based HHs are far fewer in number. To fabricate high efficient thermoelectric modules based on HH compounds, high performance p-type MNiSn systems need to be developed that are compatible with the existing n-type HH compounds. This thesis explores synthesis strategies for p-type MNiSn based compounds. In particular, the efficacy of transition metals (Sc, La) and main group elements (Al, Ga, In) as acceptor dopants on the Sn-site in ZrNiSn, was investigated by evaluating their thermoelectric performance. The most promising p-type materials could be achieved with transition metal dopants, where the introduction of Sc on the Zr side, yielded the highest Seebeck coefficient in a ternary NiSn-based HH compound up to this date. Hall effect and band gap measurements of this system showed, that the high mobility of minority carrier electrons dominate the transport properties at temperatures above 500 K. It could be shown that this is the reason, why n-type HH are successful TE materials for high temperature applications, and that p-types are subjected to bipolar effects which will lead to diminished thermoelectric efficiencies at high temperatures.rnrnTo complement the experimental investigations on different metal dopants and their influence on the TE properties of HH compounds, numerical solutions to the Boltzmann transport equation were used to predict the optimum carrier concentration where the maximum TE efficiency occurs for p-type HH compounds. The results for p-type samples showed that can not be treated within a simple parabolic band model approach, due to bipolar and multi-band effects.rnrnThe parabolic band model is commonly used for bulk TE materials. It is most accurate when the transport properties are dominated by one single carrier type. Since the transport properties of n-type HH are dominated by only one carrier type (high mobility electrons), it could be shown, that the use of a simple parabolic band model lead to a successful prediction of the optimized carrier concentration and thermoelectric efficiency in n-type HH compounds. rn

A molecular analysis of the conditional defect in MuSVts 110 viral RNA splicing

Cells infected with MuSVts110 express a viral RNA which contains an inherent conditional defect in RNA splicing. It has been shown previously that splicing of the MuSVts110 primary transcript is essential to morphological transformation of 6m2 cells in vitro. A growth temperature of 33$\sp\circ$C is permissive for viral RNA splicing,and, consequently, 6m2 cells appear morphologically transformed at this temperature. However, 6m2 cells appear phenotypically normal when incubated at 39$\sp\circ$C, the non-permissive temperature for viral RNA splicing.^ After a shift from 39$\sp\circ$C to 33$\sp\circ$C, the coordinate splicing of previously synthesized and newly transcribed MuSVts110 RNA was achieved. By S1 nuclease analysis of total RNA isolated at various times, 5$\sp\prime$ splice site cleavage of the MuSVts110 transcript appeared to occur 60 minutes after the shift to 33$\sp\circ$C, and 30 minutes prior to detectable exon ligation. In addition, consistent with the permissive temperatures and the kinetic timeframe of viral RNA splicing after a shift to 33$\sp\circ$C, four temperature sensitive blockades to primer extension were identified 26-75 bases upstream of the 3$\sp\prime$ splice site. These blockades likely reflect four branchpoint sequences utilized in the formation of MuSVts110 lariat splicing-intermediates.^ The 54-5A4 cell line is a spontaneous revertant of 6m2 cells and appears transformed at all growth temperatures. Primer extension sequence analysis has shown that a five base deletion occurred at the 3$\sp\prime$ splice site in MuSVts110 RNA allowing the expression of a viral transforming protein in 54-5A4 in the absence of RNA splicing, whereas in the parental 6m2 cell line, a splicing event is necessary to generate a similar transforming protein. As a consequence of this deletion, splicing cannot occur and the formation of the four MuSVts110 branched-intermediates were not observed at any temperature in 54-5A4 cells. However, 5$\sp\prime$ splice site cleavage was still detected at 33$\sp\circ$C.^ Finally, we have investigated the role of the 1488 bp deletion which occurred in the generation of MuSVts110 in the activation of temperature sensitive viral RNA splicing. This deletion appears solely responsible for splice site activation. Whether intron size is the crucial factor in MuSVts110 RNA splicing or whether inhibitory sequences were removed by the deletion is currently unknown. (Abstract shortened with permission of author.) ^

Dysregulation of alternative splicing of coagulation factor V results in bleeding disorder, east Texas type and other disorders of hemostasis

The studies completed herein explore different phenotypes related to the genetic defects that predispose individuals to a disruption of normal hemostasis. In the first study, a novel autosomal dominant bleeding disorder, which is characterized by excessive bleeding with trauma or surgery and menorrhagia in affected women, was studied in a large family (16 affected individuals) from east Texas. Affected members had a prolongation of their PT and/or aPTT, but normal clinical coagulation studies. Previous linkage analysis by Kuang et. al. (2001) mapped the defective gene to 1g23-24 (LODmax 7.22), which contains the gene for coagulation factor V (FV). I identified an alteration (A2440G) in the FV gene in exon 13 that segregated with the disease and was not present in 62 controls. Interestingly, this alteration resulted in a 22-fold up-regulation of a novel alternative splicing variant in patients' RNA versus controls. This translated into a similar fold increase in a 250-kDa isoform of FV seen in patients' plasma versus controls. A recombinant of this splicing event exhibited an increased sensitivity to cleavage by activated protein C (APC) that was more striking in the presence of PS. In addition, this novel isoform had increased APC cofactor activity, thus increasing the degradation of FVIIIa. These data indicated that A2440G up-regulates an alternatively spliced transcript of FV, and increases a FV isoform that hinders coagulation as opposed to promoting it like its wild-type counterpart. ^ The second study reports the largest screening to date of African Americans in two independent cohorts for a rare prothrombin variant, C20209T, which is suspected to be associated with thrombotic disease. The Texas Medical Center Genetics Resource (TexGen) Stroke DNA repository revealed 1.67% (Fisher p=0.27) of African American stroke patients were heterozygous for the 20209*T allele. Screening of the Atherosclerosis Risk in Communities Study (ARIC) cohort (n=3470) for the 20209*T allele revealed a population prevalence of 0.58% in individuals of African American descent; however, all associations with thrombotic disease were negative. Analysis of these two independent cohorts revealed that, unlike its neighbor G20210A, the C20209T variant does not increase the risk of thrombotic events in the African American population. ^

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRNP particles.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.