Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1

<p>Abstract</p> <p>Background</p> <p>Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited.</p> <p>Results</p> <p>We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective.</p> <p>Conclusions</p> <p>Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.</p

Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations

Intravenous and subcutaneous immunoglobulin preparations, consisting of IgG class antibodies, are increasingly used to treat a broad range of pathological conditions, including humoral immune deficiencies, as well as acute and chronic inflammatory or autoimmune disorders. A plethora of Fab- or Fc-mediated immune regulatory mechanisms has been described that might act separately or in concert, depending on pathogenesis or stage of clinical condition. Attempts have been undertaken to improve the efficacy of polyclonal IgG preparations, including the identification of relevant subfractions, mild chemical modification of molecules, or modification of carbohydrate side chains. Furthermore, plasma-derived IgA or IgM preparations may exhibit characteristics that might be exploited therapeutically. The need for improved treatment strategies without increase in plasma demand is a goal and might be achieved by more optimal use of plasma-derived proteins, including the IgA and the IgM fractions. This article provides an overview on the current knowledge and future strategies to improve the efficacy of regular IgG preparations and discusses the potential of human plasma-derived IgA, IgM, and preparations composed of mixtures of IgG, IgA, and IgM

The pregnancy-specific glycoprotein family of the immunoglobulin superfamily: Identification of new members and estimation of family size

The members of the carcinoembryonic antigen (CEA)/pregnancy-specific glycoprotein (PSG) gene family have a characteristic N-terminal domain that is homologous to the immunoglobulin variable region. We have estimated the size of the PSG subfamily by identification of N-domain exons from isolated genomic clones and from total genomic DNA through PCR amplification and DNA sequence determination. The PSG subfamily contains at least 11 different genes. For 7 of these, two DNA sequences differing from each other in 1 to 4 nucleotides were detected. Most likely, they represent different alleles. They are PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG11, PSG12, and PSG13. Six of the N-domain sequences described here are new. All of the PSGs except PSG1, PSG4, and PSG8 contained the arginine-glycine-aspartic acid sequence at position 93–95 corresponding to the complementarity determining region 3 of immunoglobulin. Parsimony analysis of 24 CEA and PSG sequences using 12 members of the immunoglobulin gene superfamily as out-groups to root the family free shows that the N-domain of the CEA group genes evolved in one major branch and the PSG group genes in the other

The Pregnancy-Specific Glycoprotein (PSG) Gene Cluster on Human Chromosome 19: Fine Structure of the 11 PSG Genes and Identification of 6 New Genes Forming a Third Subgroup within the Carcinoembryonic Antigen (CEA) Family

The human pregnancy-specific glycoprotein (PSG) genes belong to the carcinoembryonic antigen (CEA) family, which in turn is a member of the immunoglobulin superfamily. We have analyzed a 700-kb cosmid contig spanning the PSG region on chromosome 19q13.2. The region contains 11 closely related PSG genes organized in tandem with a highly conserved structure and organization. Seven novel genes (CGM12 to CGM18) were found in the PSG region. CGM12 belongs to the CEA subgroup and appears to be a pseudogene. CGM13 to CGM18 forms a third new subgroup within the CEA gene family. The members of this new subgroup show 94-99% identity to each other but only 70-80% to other members of either the CEA or the PSG subgroups. They are composed of exons encoding two IgC-like domains and short hydrophilic carboxyl terminals similar to those of the PSGs. Unlike any of the known CEA family genes, however, they seem to lack the exon for an IgV-like N-terminal domain

Loss of <em>Trp53</em> promotes medulloblastoma development but not skin tumorigenensis in <em>Sufu</em> heterozyhous mutant mice

Basal cell carcinoma of the skin typically carries genetic alterations in components of the hedgehog (HH) signaling pathway. Previously, we generated a knockout mouse with a loss-of-function mutation in suppressor of fused (Sufu), an essential repressor of the pathway downstream of Hh ligand cell surface reception. Mice heterozygous for the mutated Sufu allele develop a skin phenotype that includes lesions similar to basaloid follicular hamartomas. The purpose of the current study was to test the possibility that the simultaneous loss of the tumor suppressor gene, Trp53, would aggravate the Sufu skin phenotype since Trp53 loss is known to enhance the growth of other Hh-driven tumors. Consistent with previous reports, medulloblastomas and rhabdomyosarcomas developed in Sufu(+/−);Trp53(−/−) mice. However, the characteristic Sufu(+/−) skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype or proliferative capacity of the basaloid lesions. This finding was both novel and intriguing and demonstrated a differential, tissue-specific sensitivity to Sufu and Trp53 tumor suppressor gene loss, which may be linked to developmental stage and the degree of proliferative activity in specific cell types

Identification of three new genes and estimation of the size of the carcinoembryonic antigen family

Using carcinoembryonic antigen (CEA) subgroup-specific degenerate PCR primers, we have identified three new CEA gene family member L/N exons (CGM9, CGM10, and CGM11) and all previously reported L/N exons of the CEA subgroup (CEA, BGP, NCA, CGM1, CGM2, CGM6, CGM7, and CGM8). This suggests that the CEA subgroup contains 11 genes. CGM9, CGM10, and CGM11 seem to be pseudogenes. A deletion of an asparagine in CGM9 results in loss of a glycosylation site, which is conserved throughout the CEA gene family. We have previously suggested the number of genes in the pregnancy-specific glycoprotein (PSG) subgroup to be 11, which together with this study indicates that the CEA gene family contains 22 genes in all. Parsimony analysis of the CEA subgroup interrelationships suggests that CGM7 occupies the most primitive position within the CEA subgroup, being a sister group to the rest. CEA, BGP, NCA, and CGM1 form a fairly well-supported group within the CEA subgroup

A small amphipathic α-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5

Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and acti– vators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affected Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine-phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine-phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, we have observed that tyrosine-phosphorylated carboxyl-truncated forms of Stat5 proteins were considerably more stable than phos– phorylated wild-type forms of the protein. Also, the C–terminal region of Stat5 could confer proteasome-dependent downregulation to Stat1. With a series of C–terminal deletion mutants, we have defined a relatively small, potentially amphipathic α-helical region that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome-dependent pathway

Inhibition of GLI1 gene activation by Patched1

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via ‘negative feedback’ is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180–786 and 1058–1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling