Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.     

A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop

The mouse D3 dopamine receptor has been cloned from olfactory tubercle cDNA using polymerase chain reaction and has been found to exist in two alternatively spliced forms. These two mRNA isoforms differ by the presence or absence of 63 base pairs (bp), which encode 21 amino acids in the putative third cytoplasmic loop of the receptor. The longer form corresponds to the previously reported rat D3 dopamine receptor, to which it bears sequence homology of 94%. Northern blot analysis shows the mouse D3 receptor to be most abundant in the olfactory tubercle. Expression studies show the novel short D3 isoform to bind dopaminergic ligands with a D3-like pharmacological profile. Polymerase chain reaction analysis on different mouse brain regions shows the long and short D3 receptors to be present in the same tissues, the longer form invariably being the predominant one. Analysis of the gene for the mouse D3 dopamine receptor shows that no separate exon encodes the 63-bp stretch and reveals the presence of a consensus sequence for an acceptor site at the 3' end of the 63-bp stretch. This suggests that an internal acceptor site in the exon coding for the distal part of the third cytoplasmic loop directs alternative splicing of the D3 dopamine receptor.     

Axonal injury alters alternative splicing of the retinal NR1 receptor: the preferential expression of the NR1b isoforms is crucial for retinal ganglion cell survival

Cellular-specific splicing of the retinal NMDAR1 receptor (NR1) and expression of NMDAR2 receptor (NR2) subunits in response to optic nerve injury was investigated by in situ hybridization in adult rats. A controlled optic nerve crush led to a clear alteration in the expression of alternatively spliced NR1 variants in the retinal ganglion cell layer (GCL). The NR1-2b and NR1-4b isoforms were preferentially expressed between 2 d and 1 week after injury, whereas expression for all other isoforms remained either unchanged or decreased to barely detectable levels within 4 weeks. Cellular silver grain density for NR2 subunits also declined in the GCL after trauma. To directly test the hypothesis that NR1b expression is crucial for cell survival after axonal trauma, we administered intraocularly an antisense oligonucleotide against the NR1b isoform 2 and 3 d after injury. This led to a drastic loss of retrogradely labeled retinal ganglion cells (RGCs). Antisense targeting clearly reduced retinal NR1 protein levels, as judged by Western blot analysis, but had no effect on the cell number in control retinas. These findings point toward injury-specific changes in alternative splicing of the NR1 receptor, which are crucial for the survival of RGCs after partial axonal trauma. We therefore propose that this reflects an adaptive, rather than a pathogenic, cellular response to neurotrauma.     

Fli-1在肿瘤发生及发展中的作用

Friend白血病病毒插入位点1(Fli-1)是属于E26转化特异性因子(ETS)家族的转录因子,在造血干细胞、血管内皮细胞、淋巴细胞和恶性肿瘤细胞中表达,在血管内皮细胞生成和肿瘤细胞增殖过程中具有重要调节作用.血管内皮细胞不仅是正常血管生成的关键因素,而且在肿瘤的生长和转移中均需要新生血管的形成作为肿瘤进展的驱动因素.以Fli-1为治疗靶点,既可抑制肿瘤细胞的增殖,又可阻断肿瘤血管的形成,将成为治疗肿瘤的优秀靶点.因此,详细研究Fli-1结构与功能及其在恶性肿瘤中的表达情况,有助于进一步明确Fli-1在恶性肿瘤发生及发展中的作用.本文作者就Fli-1与肿瘤关系的研究进行综述.     

Induction of alternative splicing of HLA-B27 by bacterial invasion

This study introduces an attributional processing approach to study age differences in dispositional attributions. Dispositional attributions made in the context of relationship vignettes were examined among younger and older adults in 2 conditions (immediate and delayed rating conditions). By using a direct assessment of a 2-step process for making dispositional attributions, it was inferred that a spontaneous adjustment stage occurred following an initial characterization stage as a function of age group and content of vignettes. Older adults made lower dispositional ratings if they were given more time to think about the situations than if asked to make an immediate judgment. By contrast, younger adults made higher dispositional ratings if they were given more time to make the judgments. Qualitative analyses of schemas elicited by a subsample of participants for each vignette suggested a relationship between dispositional attributional ratings and content-evoked schemas.     

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.     

Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.