Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.     

Cyclooxygenase variants: the role of alternative splicing

Alternative splicing of cellular pre-mRNA is responsible for production of multiple mRNAs from individual genes. Splice variants are expressed in cell- and tissue-specific contexts that are important in development and physiology. Alternative splicing can serve as a regulatory mechanism whereby developmental programming and environmental factors/stimuli affect biological activities of translated proteins. Cyclooxygenase (COX)-1 and -2 genes produce splice variants whose biological expression, relevance, and activities have been of significant interest. COX variants are produced by a variety of splicing mechanisms. Four structural domains of the COX proteins (the amino terminal signal peptide, membrane-binding domain, dimerization domain, and catalytic domain) are defined by specific COX exons. COX splice variants may, therefore, result in potential changes in protein subcellular localization, dimerization, and activity. COX variant proteins may act in roles which diverge from those of COX-1 and -2.     

Detection and measurement of alternative splicing using splicing-sensitive microarrays

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

ASD及其在基因选择性剪接检索中的应用

在生物信息学的飞速发展中,与之相应的各种类型的数据库不断涌现,选择性剪接数据库（Alternative Splicing Database）便是其中之一。本文详细介绍了ASD数据库的主要内容及其功能,并在其子数据库AltSplice中检索NGAL基因的选择性剪接,由此为例说明了ASD数据库在基因选择性剪接检索中的应用。     

New Species of Human Tyrosine Hydroxylase mRNA Are Produced in Variable Amounts in Adrenal Medulla and Are Overexpressed in Progressive Supranuclear Palsy

Abstract: Alternative splicing of human tyrosine hydroxylase (TH) pre-mRNA produces four mRNAs leading to four different TH isoforms and is thought to have important regulatory functions. We show that the diversity of TH mRNAs is greater than previously described in the autonomous nervous system: New splice junctions corresponding to the skipping of exon 3 were identified by amplification of cDNA synthesized from pheochromocytoma RNA. In all cases the reading frame was maintained. These species were assayed by RNase protection experiments; their abundance (4–6%) was comparable to that of the previously identified human TH-3 and -4 species in normal adrenal medulla. However, higher levels (11–34%) of these species were found in adrenal medullas of patients suffering from progressive supranuclear palsy. Whether such changes are specific to the disease or the consequences of the stress associated with this severe neurodegeneration remains to be established.     

Alternative splicing of human and mouse NPFF2 receptor genes: Implications to receptor expression

Alternative splicing has an important role in the tissue-specific regulation of gene expression. Here we report that similar to the human NPFF2 receptor, the mouse NPFF2 receptor is alternatively spliced. In human the presence of three alternatively spliced receptor variants were verified, whereas two NPFF2 receptor variants were identified in mouse. The alternative splicing affected the 5′ untranslated region of the mouse receptor and the variants in mouse were differently distributed. The mouse NPFF system may also have species-specific features since the NPFF2 receptor mRNA expression differs from that reported for rat.     

Surface expression and distribution of voltage-gated potassium channels in neurons (Review)

The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.     

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.