The G-->A mutation at position 20210 of the prothrombin or coagulation factor II gene (F2) represents a common genetic risk factor for the occurrence of thromboembolic events. This mutation affects the 3'-terminal nucleotide of the 3' untranslated region (UTR) of the mRNA and causes elevated prothrombin plasma concentrations by an unknown mechanism. Here, we show that the mutation does not affect the amount of pre-mRNA, the site of 3' end cleavage or the length of the poly(A) tail of the mature mRNA. Rather, we demonstrate that the physiological F2 3' end cleavage signal is inefficient and that F2 20210 G-->A represents a gain-of-function mutation, causing increased cleavage site recognition, increased 3' end processing and increased mRNA accumulation and protein synthesis. Enhanced mRNA 3' end formation efficiency emerges as a novel principle causing a genetic disorder and explains the role of the F2 20210 G-->A mutation in the pathogenesis of thrombophilia. This work also illustrates the pathophysiologic importance of quantitatively minor aberrations of RNA metabolism.

The functional analysis of the common prothrombin 20210 G>A (F2 20210*A) mutation has recently revealed gain of function of 3end processing as a novel genetic mechanism predisposing to human disease. We now show that the physiologic G at the cleavage site at position 20210 is the functionally least efficient nucleotide to support 3end processing but has evolved to be physiologically optimal. Furthermore, the F2 3end processing signal is characterized by a weak downstream cleavage stimulating factor

Numerous cellular mRNAs encoding proteins critical during cell stress, apoptosis, and the cell cycle seem to be translated by means of internal ribosome entry sequences (IRES) when cap-dependent translation is compromised. The underlying molecular mechanisms are largely unknown. Using a HeLa-based cell-free translation system that mirrors the function of cellular IRESs in vitro, we recently demonstrated that translation from the c-myc IRES continues after proteolytic cleavage of eukaryotic translation initiation factor (eIF) 4G. To address the role of eIF4G in cellular IRES-driven translation directly, we immunodepleted eIF4GI from the HeLa cell translation extracts. After efficient depletion of eIF4GI (>90%), both cap-dependent and c-myc IRES-dependent translations are diminished to residual levels (<5%). In striking contrast to cap-dependent translation, c-myc IRES-dependent translation is fully restored by addition of the conserved middle fragment of eIF4GI, harboring the eIF3-and eIF4A-binding sites. p97, an eIF4G-related protein that has been described both as an inhibitor of translation and as a modulator of apoptosis, not only suffices to also rescue c-myc IRES-driven (but not cap-dependent) translation, but it even superinduces IRES-mediated translation 3-fold compared with nondepleted extracts. Interestingly, both p97 and the middle fragment of eIF4GI also rescue translation driven by proapoptotic (p97) and antiapoptotic [X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1 (c-IAP1)] IRESs, reflecting a broader role of these polypeptides in cellular IRES-mediated translation and indicating their importance in apoptosis. For most eukaryotic mRNAs, translation initiation involves binding of the small ribosomal subunit to the capped 5Ј end of the mRNA followed by scanning to the start codon. The eukaryotic initiation factor (eIF) 4G plays a key role in capmediated ribosome recruitment. The function of eIF4G includes circularization of the mRNA by interaction with the cap-binding protein eIF4E and the poly(A)-binding protein (PABP), delivery of the RNA-helicase eIF4A to the 5Ј end of the mRNA, and bridging of mRNA and 40S ribosomal subunit by means of eIF3 (1-3).Internal ribosomal entry does not require a 5Ј 7 mGpppN cap-structure (4, 5) and has been described as an alternative mechanism of translation initiation for many viral RNAs and an increasing number of cellular mRNAs (6, 7). Interestingly, many of these cellular mRNAs encode proteins involved in important cellular processes, including development, cell cycle, apoptosis, wound repair, and angiogenesis (8-15).The mRNA encoding the transcription factor c-myc can be translated by means of an internal ribosomal entry sequence (IRES) in its 5Ј UTR (16,17). c-myc plays a critical role in the control of both cell proliferation͞differentiation and apoptosis. Deregulation of c-myc expression is associated with a wide range of hematopoietic neoplasms, and the c-myc IRES has been implicated in the development of multiple myeloma (18,19)....

Pediatric HL patients with a negative PET in response assessment have an excellent prognosis while PET-positive patients have an increased risk for relapse.

The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.

The clinical course of neuroblastoma is more heterogeneous than any other malignant disease. Most low-risk patients experience regression after limited or even no chemotherapy. However, more than half of high-risk patients die from disease despite intensive multimodal treatment. Precise patient characterization at diagnosis is key for risk-adapted treatment. The guidelines presented here incorporate results from national and international clinical trials to produce recommendations for diagnosing and treating neuroblastoma patients in German hospitals outside of clinical trials.

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Despite intensive treatment regimens, high-risk and late-stage neuroblastoma tends to have a poor survival outcome. Overexpression of the apoptotic regulator, X-linked inhibitor of apoptosis protein (XIAP), has been associated with chemotherapy resistance in several cancers including neuroblastoma. Here, we report preclinical evidence that XIAP offers an effective therapeutic target in neuroblastoma. Human and murine neuroblastoma cells were treated with the Smac mimetic LBW242 alone or in combination with cytotoxic drugs used clinically to treat neuroblastoma. Expression of XIAP protein, but not mRNA, was highly increased in neuroblastoma cells compared to healthy adrenal gland tissue, consistent with a posttranscriptional regulation of XIAP expression. Treatment with LBW242 sensitized human and murine neuroblastoma cells to chemotherapy-induced apoptosis, which was mediated by activation of both the intrinsic and extrinsic apoptosis pathways. Although Smac mimetics have been reported to stimulate TNF-a-induced apoptosis by degradation of cellular IAP (cIAP)-1/2, we found that LBW242-mediated sensitization in neuroblastoma cells occurred in a TNF-a-independent manner, despite induction of cIAP-1/2 degradation and TNF-a expression. Together, our findings show that XIAP targeting sensitizes neuroblastoma to chemotherapy-induced apoptosis, suggesting a novel therapeutic approach to treat this childhood malignancy. Cancer Res; 72(10); 2645-56. Ó2012 AACR.