Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities

An intronic hexanucleotide UGCAUG has been shown to play a critical role in the regulation of tissue-specific alternative splicing of pre-mRNAs in a wide range of tissues. Vertebrate Fox-1 has been shown to bind to this element, in a highly sequence-specific manner, through its RNA recognition motif (RRM). In mammals, there are at least two Fox-1-related genes, ataxin-2 binding protein 1 (A2BP1)/Fox-1 and Fxh/Rbm9, which encode an identical RRM. Here, we demonstrate that both mouse Fxh and A2BP1 transcripts undergo tissue-specific alternative splicing, generating protein isoforms specific to brain and muscle. These tissue-specific isoforms are characterized for their abilities to regulate neural cell-specific alternative splicing of a cassette exon, N30, in the non-muscle myosin heavy chain II-B pre-mRNA, previously shown to be regulated through an intronic distal downstream enhancer (IDDE). All Fxh and A2BP1 isoforms with the RRM are capable of binding to the IDDE in vitro through the UGCAUG elements. Each isoform, however, shows quantitative differences in splicing activity and nuclear distribution in transfected cells. All Fxh isoforms and a brain isoform of A2BP1 show a predominant nuclear localization. Brain isoforms of both Fxh and A2BP1 promote N30 splicing much more efficiently than do the muscle-specific isoforms. Skeletal muscles express additional isoforms that lack a part of the RRM. These isoforms are incapable of activating neural cell-specific splicing and, moreover, can inhibit UGCAUG-dependent N30 splicing. These findings suggest that tissue-specific isoforms of Fxh and A2BP1 play an important role in determining tissue specificity of UGCAUG-mediated alternative splicing

Immunological Detection and Characterization of Poly(A) Polymerase, Poly(A)-Binding Protein and Cytoplasmic Polyadenylation Element-Binding Protein in Goldfish and Xenopus Oocytes

Cytoplasmic polyadenylation regulates translational activation of dormant cyclin B1 mRNA stored in immature oocytes, a process required for the initiation of oocyte maturation in goldfish and Xenopus. As a first step towards understanding the molecular mechanisms of translational activation of cyclin B1 during oocyte maturation, we have isolated cDNA clones encoding proteins involved in cytoplasmic polyadenylation and produced specific antibodies against recombinant proteins. These include poly(A) polymerase (PAP), poly(A)-binding protein (PABP) and cytoplasmic polyadenylation element-binding protein (CPEB). Monoclonal antibodies raised against goldfish PAP recognized several forms of PAP in goldfish and Xenopus oocytes. Besides ordinary PAPs with high molecular weight (ca. 100 kDa), the antibodies also detected those with low molecular weight (ca. 40 kDa) that are present specifically in the cytoplasm, raising new players that might be responsible for cytoplasmic polyadenylation. An antibody against goldfish PABP showed for the first time in Xenopus oocytes the protein expression of PABPII, another PABP distinct from the well-characterized PABPI. Monoclonal antibodies raised against Xenopus CPEB recognized both unphosphorylated 62-kDa and phosphorylated 64-kDa forms but did not cross-react with goldfish CPEB, which was specifically detected by anti-goldfish CPEB monoclonal antibodies produced previously. The cDNAs, recombinant proteins and antibodies produced in this study are expected to provide useful tools for investigating the regulatory mechanisms of cyclin B1 translation during oocyte maturation in goldfish and Xenopus

Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-κB

Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is identified as a novel cell surface marker for human T-cell leukemia virus (HTLV-1)-infected T cells. Adult T-cell leukemia/lymphoma (ATLL) is developed in HTLV-1-infected T-cells after a long infection period. To examine the mechanism of CADM1 overexpression in ATLL, we first identified that CADM1 is transcriptionally up-regulated by a transcriptional enhancer element through NF-κB signaling pathway. In HTLV-1-infected T-cells, CADM1 expression is dependent on HTLV-1/Tax through activation of canonical and non-canonical NF-κB; however, in ATLL cells with frequent loss of Tax expression, the activation of canonical NF-κB only enhances the CADM1 expression. Along with active mutations in signaling molecules under T-cell recepor (TCR) signaling, degradation of p47, a negative regulator of NF-κB, was essential for activation of canonical NF-κB through stabilization of NEMO (NF-κB essential modulator). The mechanism of p47 degradation is primarily dependent on activation of lysosomal-autophagy and the autophagy is activated in most of the HTLV-infected and ATLL cells, suggesting that the p47 degradation may be a first key molecular event during HTLV-1 infection to T-cells as a connector of two important signaling pathways, NF-κB and autophagy

Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2^low Adult T-Cell Leukemia/Lymphoma

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL

Critical Role for TSLC1 Expression in the Growth and Organ Infiltration of Adult T-Cell Leukemia Cells In Vivo▿

Adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 infection. The tumor suppressor lung cancer 1 (TSLC1) gene was previously identified as a novel cell surface marker for ATL, and this study demonstrated the involvement of TSLC1 expression in tumor growth and organ infiltration of ATL cells. In experiments using NOD/SCID/γcnull mice, both leukemia cell lines and primary ATL cells with high TSLC1 expression caused more tumor formation and aggressive infiltration of various organs of mice. Our results suggest that TSLC1 expression in ATL cells plays an important role in the growth and organ infiltration of ATL cells