Identification of novel splice variants of the Arabidopsis DCL2 gene

In Arabidopsis thaliana, Dicer-like protein 2 (DCL2) cleaves double-stranded virus RNA, playing an essential role in the RNA interference pathway. Here, we describe three alternative splicing (AS) forms of AtDCL2: in one, both intron 8 and intron 10 are retained in the mRNA, in second only intron 8 is retained and in the third no intron is retained, but there is a deletion of 56 nucleotides at the end of exon 10. These splicing forms are present in stems and leaves at different development stages. AS was also detected in DCL2 of Brassica rapa, where intron 9, but not intron 8 or intron 10, was retained suggesting that AS may be a common phenomenon in cruciferous plant DCL2s. The retained introns and sequence deletions detected in AtDCL2 changed the reading frame and produced premature terminal codons. The AS forms appeared to be substrates of nonsense-mediated decay of mRNA. Fei Yan and Jiejun Peng contributed equally to this work.     

Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.     

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.     

Collecting splice variants

A DNA array has been used to measure the affinity of wild-type and mutant variants of a zinc-finger-protein DNA-binding domain for all combinations of its binding site on DNA.

     

PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants

The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.     

Expression of OB receptor splice variants during prenatal development of the mouse

In adult animals, signaling through the leptin receptor (OB-R) has been shown to play a critical role in fat metabolism. However, it is not known when these receptors are first expressed and what their role may be during embryonic development. To date, at least 6 splice variants of the OB-R have been identified. Although the function of each of these individual splice variants are unknown, only one of them, ob-rL ,encodes a receptor with a long intracellular domain that is implicated in OB-R signaling. In this study we have used in situ hybridization to examine the localization of OB-R splice variants during embryonic development of C57B1/6J mice. Using a probe, ob-r, that recognizes all of the splice variants, ob-r mRNA was found to be distributed in developing bone, mesenchyme, notochord and liver. In addition, epithelial structures including leptomeninges, choroid plexi and hair follicles also expressed ob-r. No ob-r mRNA was detected in the CNS. ob-rL, expression was only detected in notochord, bone and mesenchyme. The differential expression of these two mRNA isoforms suggests that the extracellular and intracellular domains of the OB receptor perform different biological functions.     

猪PILRA基因克隆及变异剪接体鉴定

Ⅱ型成对免疫球蛋白样受体(Paired immunoglobulin-like type 2 receptors, PILRs)是免疫球蛋白超家族成员之一,包括α和β两个亚型。PILRα在机体抵抗病原体入侵的免疫反应中发挥着重要作用,但目前尚未见关于猪PILRα的报道。为了分析其在猪抗病育种中的作用,本文克隆了猪PILRα编码基因(PILRA)并鉴定变异剪接体,利用实时荧光定量PCR方法构建其组织表达谱和诱导表达谱。结果表明,成功克隆了猪PILRA基因的3个变异剪接体V1~V3(GenBank登录号：KJ143679~81),预测的蛋白质多肽链分别长271 aa、254 aa和283 aa,且都具有免疫球蛋白结构域。各变异剪接体均在脾脏中表达量最高,肝脏、肺脏次之,在心脏、肾脏、胃、肌肉、淋巴、大肠、小肠和膀胱等组织中表达量较低或检测不到。Poly(I:C)能显著诱导变异剪接体V1的表达,但几乎不影响V2、V3的表达。研究结果为进一步揭示PILRA在猪抗病育种中的作用提供了基础。     

Identification of novel splice variants of the human CD44 gene

The CD44 gene contains 10 variable exons (v1-v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms, several of which have been implicated in the metastatic spread of tumour cells. Here, we describe a cryptic splice site, in intron 6 of the human CD44 gene, used during mRNA processing. This cryptic splice site is used in conjunction with variable exon 3, or independently from it in the form of a pseudo-exon of 49 bp, which generates a stop codon by frame shift in the contiguous variable exon downstream. This pseudo-exon has been found inserted immediately 3' to any other variable exon from v4 to v10, in the final CD44 mRNA. The implication of this cryptic splice site in haltering CD44 protein translation is questioned in the context of Nonsense Mediated Decay and the overall regulation of CD44 expression.     

Functional characterization and identification of mouse Rad51d splice variants

Background  

The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns.     

Expression of mouse 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA alternative splice variants in hypoxia

Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) mRNA alternative splice variants was studied in different mouse tissues in hypoxic conditions in vivo. Significant increase of the expression of PFKFB-3 mRNA was observed in the mouse lungs, testes and brain in hypoxia. Several new alternative splice variants of PFKFB-3 mRNA were identified in the lung, testis, brain and skeletal muscle. They have different length and amino acid sequence of C-terminal regulatory part. However, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase catalytic domains were identical. Moreover, the expression of different alternative splice variants of PFKFB-3 mRNA has shown tissue specificity and different levels of induction in hypoxic conditions in vivo. Results of this investigation indicate a possible role of PFKFB-3 splice isoform in cell adaptation to hypoxic conditions.     

NR2A and NR2B receptor gene variations modify age at onset in Huntington disease in a sex-specific manner

In addition to the pathogenetic CAG repeat expansion other genetic factors play a significant role in determining age at onset (AO) in Huntington disease (HD), e.g. variations in the NR2A and NR2B glutamate receptor subunit genes (GRIN2A, GRIN2B). In order to expand these findings we fine-mapped a larger HD patient panel (n = 250) using densely spaced markers flanking the originally associated SNPs in GRIN2A and GRIN2B. In GRIN2A association fine-mapping based on eight additional SNPs confirmed intron 2 as the region of strongest association. In GRIN2B fine-mapping with seven additional SNPs consolidated C2664T as causal genetic variation. Gender stratification of patients revealed differences in the variability in AO attributable to the CAG repeat number and highly significant differences in the AO association with the C2664T and rs8057394/ rs2650427 variations. Addition of the corresponding genotype variations to the effect of CAG repeat lengths resulted in a significant increase of the R ² values only in females. The sex-specific effect for C2664T is underscored by differences in the genotype and allele frequencies observed for female versus male HD patients (P = 0.01) caused by decreased CC frequency in females. Overall, female HD patients homozygous for the CC genotype tended to have later AO compared to the other two genotypes. Stratification of the results by presumed menopausal status demonstrated that the significant findings were predominantly observed in pre-menopausal patients. We speculate that altered hormone levels herald protective effects of this genotype. Together, GRIN2A and GRIN2B genotype variations explain 7.2% additional variance in AO for HD.     

Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants

In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2 alpha R (443 aa) and mOX2 beta R (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2 alpha R, and the mOX2 beta R revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP(3)) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTP gamma S revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP(3) production at all concentrations in the mOX2 beta R variant. Orexin-A, however, showed no difference in IP(3) production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2 alpha R mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2 beta R gene expression, compared with both mOX1R and mOX2 alpha R. This potentially implicates a fundamental physiological role for these splice variants.