Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5 or the 3 adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected M r are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.In trans-splicing, two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process is believed to proceed through two trans-esterification steps that result in the linking of the two exons by a normal 3Ј-5Ј phosphodiester bond. This process has been described mostly in trypanosoma, nematodes, plant͞algal chloroplasts, and plant mitochondria (1).Trans-splicing of artificial pre-mRNAs in mammalian cells in vitro has been reported but with some limitations (2-5). In addition, spliced leader RNAs from nematodes or from Simian virus 40 can be accurately trans-spliced in transfected COS cells, which reveals functional conservation in the splicing machinery between lower eukaryotes and mammals and demonstrated the potential for trans-splicing in mammalian cells (6). Studies in vitro also have shown that a synthetic pre-mRNA substrate containing an exon and a 5Ј donor splice site can be efficiently trans-spliced to another synthetic pre-mRNA (3Ј trans-splicing substrate) if this contains either exonic enhancers or a downstream 5Ј splice site (7-8). Several examples of possible natural trans-splicing in mammalian cells have been reported (9-12), but none of these trans-splicing have been demonstrated in vitro.During our current investigation on the carnitine octanoyltransferase (COT) gene, that encodes for an enzyme, which transports medium-chain fatty acids through the peroxisome, we isolated a cDNA COT clone, which had exon 2 repeated. We report here that the pre-mRNA of COT from rat liver produces...

The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P ‫؍‬ .04) and a shorter diseasefree survival (P ‫؍‬ .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre-miR135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a-mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients. (Blood. 2009;114:2945-2951)

IntroductionZAP-70 protein is a 70-kDa member of the Syk family of protein tyrosine kinases that was first identified as a crucial element for proximal signaling from the T-cell receptor. 1 Similar to Syk protein after the B-cell receptor (BCR) stimulation, ZAP-70 is recruited to the phosphorylated immunoreceptor tyrosine activation motifs of the -and CD3-chains present in the T-cell receptor, where it subsequently becomes phosphorylated and initiates several signaling cascades. 2 Expression of ZAP-70 protein was considered to be restricted to T lymphocytes and natural killer cells. However, it has also been found expressed in normal B-cell precursors and in some subsets of activated B cells. [3][4][5][6] Among B cell-derived malignancies, ZAP-70 is mainly expressed in chronic lymphocytic leukemia (CLL; 37%-57% of cases), 7-9 B-acute lymphoblastic leukemia (B-ALL; 56%-59% of cases) 4,10 and Burkitt lymphoma (8%-31% of cases). 11,12 An increased ZAP-70 expression in CLL has been associated with particular adverse biologic features, such as the presence of unmutated IgHV genes 7 or high CD38 expression, 13 and correlates with a poor clinical outcome. [7][8][9] In the same vein, ZAP-70 expression in B-ALL correlates with a short survival as well. 10 Although ZAP-70 expression in B-cell malignancies has an adverse prognostic influence, its role in the biology of the tumoral B cell is not fully defined. In this regard, the expression of ZAP-70 protein in CLL cells has been related to an enhanced BCR signaling. [13][14][15][16][17] In addition, increased ZAP-70 expression has been associated with increased migration capabilities of CLL cells toward different chemokines, such as CXCL12, CCL19, and CCL21, [18][19][20] and with increased signaling and survival on CXCL12 treatment. 18,21 However, whether these increased migrative capabilities are a direct effect of ZAP-70 expression or a mere reflection of the distinct biology features of ZAP-70-expressing cells needs to be further investigated.To ascertain the direct implication of ZAP-70 in B-cell signaling and migration, we analyzed the phenotypic effects of ectopic ZAP-70 expression in a B-cell system and studied the expression of adhesion molecules and chemokine receptors in CLL primary cells with high or low ZAP-70 expression within the same patient. Herein, we report that IgM, but not IgD, stimulation mobilizes and activates ZAP-70, which in turn enhances BCR-induced ERK1/2 and Akt phosphorylation and delays IgM and CD79b internalization. Moreover, we show that ZAP-70 induces the expression of the chemokine receptor CCR7 via ERK1/2 activation, thus directly enhancing the capacity of signaling and migration of the ZAP-70-expressing B cells toward CCL21. Finally, we show that CLL cells with higher ZAP-70 expression within the same patient have a different expression profile of adhesion molecules and chemokine receptors and enhanced migration capacity toward CCL21. Methods Cell lines and primary cellsThe Burkitt lymphoma B-cell lines Raji and Ramos were obtained from ...