Evidence for a novel splice variant of the alpha subunit of Go in rat male haploid germ cells.

Northern analysis shows that Go alpha mRNA is highly expressed in immature germ cells from rat. Whereas alpha o2 mRNA is the major form in pachytene spermatocytes, a message of shorter chain length is present in large amount in haploid germ cells. This mRNA was detected with an oligonucleotide specific for the 3'-coding region of alpha o2, but did not hybridize to oligonucleotides specific for the 5'-untranslated and 5'-coding regions. The results indicate the presence of a novel splice variant of alpha o mRNA, which may code for a Go alpha protein important for germ cell development.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Alpha1 soluble guanylyl cyclase (sGC) splice forms as potential regulators of human sGC activity

Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one alpha- and one beta-subunit. The alpha(1)/beta(1) sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new alpha(1) sGC protein variants generated by alternative splicing. The 363 residue N1-alpha(1) sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-alpha(1) sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-alpha(1) sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-alpha(1), N2-alpha(1) sGC mRNA levels together with RT-PCR analysis for C-alpha(1) sGC demonstrated that the expression of the alpha(1) sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-alpha(1) sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the alpha(1)/beta(1) heterodimer. The C-alpha(1) sGC variant heterodimerizes with the beta(1) subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-alpha(1) band did not change in response to ODQ treatments, while the level of 83 kDa alpha(1) band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues.     

Study of GOLPH3: a Potential Stress-Inducible Protein from Golgi Apparatus

Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.     

A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines

We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages). Three other presumed splice variant isoforms have also been identified for MKP-M. The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein. The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined. MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) > p38 = extracellular signal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages.     

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated 'rpL22-like short') that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.     

Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): a possible modulator of nucleotidase activity and purinergic signaling

Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3beta", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3alpha". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3alpha, the alternatively spliced NTPDase3beta was expressed in COS cells after transfection, but only the full-length NTPDase3alpha is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3beta was co-transfected with full-length NTPDase3alpha, there was a significant reduction in the amount of NTPDase3alpha that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3beta variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3alpha and NTPDase3beta.     

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.