ZBP1 (zipcode-binding protein 1) was originally discovered as a trans-acting factor for the ''zipcode'' in the 39 untranslated region (UTR) of the b-actin mRNA that is important for its localization and translational regulation. Subsequently, ZBP1 has been found to be a multifunctional regulator of RNA metabolism that controls aspects of localization, stability, and translation for many mRNAs. To reveal how ZBP1 recognizes its RNA targets, we biochemically characterized the interaction between ZBP1 and the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element located within the first 28 nucleotides of the zipcode. The spacing between the RNA sequences is consistent with the structure of IMP1 KH34, the human ortholog of ZBP1, that we solved by X-ray crystallography. The tandem KH domains are arranged in an intramolecular anti-parallel pseudodimer conformation with the canonical RNA-binding surfaces at opposite ends of the molecule. This orientation of the KH domains requires that the RNA backbone must undergo an~180°change in direction in order for both KH domains to contact the RNA simultaneously. The RNA looping induced by ZBP1 binding provides a mechanism for specific recognition and may facilitate the assembly of posttranscriptional regulatory complexes by remodeling the bound transcript.[Keywords: ZBP1; RNA-binding protein; KH domain; RNA localization] Supplemental material is available at http://www.genesdev.org.

Treatments for immune thrombocytope-nic purpura (ITP) providing durable plate-let responses without continued dosing are limited. Whereas complete responses (CRs) to B-cell depletion in ITP usually last for 1 year in adults, partial responses (PRs) are less durable. Comparable data do not exist for children and 5-year outcomes are unavailable. Patients with ITP treated with rituximab who achieved CRs and PRs (platelets > 150 10 9 /L or 50-150 10 9 /L, respectively) were selected to be assessed for duration of their response; 72 adults whose response lasted at least 1 year and 66 children with response of any duration were included. Patients had baseline platelet counts < 30 10 9 /L; 95% had ITP of > 6 months in duration. Adults and children each had initial overall response rates of 57% and similar 5-year estimates of persisting response (21% and 26%, respectively). Children did not relapse after 2 years from initial treatment whereas adults did. Initial CR and prolonged B-cell depletion predicted sustained responses whereas prior splenectomy, age, sex, and duration of ITP did not. No novel or substantial long-term clinical toxicity was observed. In summary, 21% to 26% of adults and children with chronic ITP treated with standard-dose rituximab maintained a treatment-free response for at least 5 years without major toxicity. These results can inform clinical decision-making. (Blood. 2012;119(25):5989-5995)

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 59 element (CGGAC) followed by a variable 39 element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

IntroductionImmune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by production of autoreactive antibodies to platelet antigens, resulting in both accelerated destruction of platelets and reduced platelet production. 1 While healthy individuals harbor platelet-specific autoreactive T cells that are tolerized in the periphery, 2 patients with ITP possess activated platelet autoreactive T cells and cytokine imbalance, 3-7 suggesting loss of peripheral tolerance in ITP patients. CD4 ϩ regulatory T cells (Tregs) play an important role in maintenance of peripheral tolerance and are characterized by the expression of the CD25 surface marker and the transcription factor forkhead box protein 3 (Foxp3), making up 5% to 10% of the normal CD4 ϩ T-cell population. 8 Different populations of Tregs have been described, including naturally occurring and inducible Tregs. 9 The former are thymically derived and suppress general autoreactive responses under noninflammatory conditions, although they can also become activated and expand in an antigen-specific manner. 10 Inducible Tregs are generated in the periphery through exposure to antigen, but once activated are thought to mediate suppressive activity against other antigens by the local release of specific cytokines. 11 Several reports have demonstrated Treg alterations in a number of autoimmune diseases. [12][13][14][15][16] These reports suggest that circulating Treg frequency and/or function may be used as a marker for evaluating autoimmune status in patients. Recent studies in patients with ITP have shown reduced levels of Foxp3 mRNA 17 and protein 18 in circulating mononuclear cells and abnormal Treg function in spleen biopsies. 19 These studies indicate that deficiency in generation and/or defective functions of Tregs may contribute to loss of immunologic self-tolerance in patients with ITP. To test the hypothesis that the pathogenesis of chronic ITP may be related to the levels or function of circulating peripheral Tregs, we examined the frequency of Tregs in peripheral blood mononuclear cells (PBMCs) from patients with chronic ITP by flow cytometry and performed in vitro assays to assess the immunosuppressive effect of Tregs on CD4 ϩ T-cell proliferation. Methods SubjectsWe enrolled 17 patients with chronic refractory ITP (Table 1) and 16 age-matched and closely age-matched healthy donors in this study, and informed consent was obtained in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Boards of the Weill Medical College of Cornell University and of the New York Blood Center (NYBC). Cell staining and purificationWithin 2 hours of collection, whole blood was stained with anti-CD4 and anti-CD25 (both from BD Pharmingen, San Diego, CA) followed by Foxp3 staining (clone PCH101; eBioscience, San Diego, CA) according to the manufacturer's instructions and analyzed by flow cytometry (FACSCanto cytometer with FACSDiva software; BD Biosciences, San Jose, CA). Due to the lack of a Treg cell-specific surface ma...

Adults with newly diagnosed or persistent immunothrombocytopenia frequently relapse upon tapering steroids; adults and children with chronic disease have an even lower likelihood of lasting response. In adults with newlydiagnosed immunothrombocytopenia, two studies showed that dexamethasone 40 mg/day x four days and 4 rituximab infusions were superior to dexamethasone alone. Studies have also shown three cycles of dexamethasone are better than one and patients with persistent/chronic immunothrombocytopenia respond less well to either dexamethasone or rituximab. Therefore, 375 mg/m 2 x 4 rituximab was combined with three 4-day cycles of 28 mg/m 2 (max. 40 mg) dexamethasone at 2-week intervals and explored in 67 ITP patients. Best long-term response was assessed as complete (platelet count ≥100x10 9 /L) or partial (50-99x10 9 /L). Only 5 patients had not been previously treated. Fifty achieved complete (n=43, 64%) or partial (n=7, 10%) responses. Thirty-five of 50 responders maintained treatment-free platelet counts over 50x10 9 /L at a median 17 months (range 4-67) projecting 44% eventfree survival. Duration of immunothrombocytopenia less than 24 months, achieving complete responses, and being female were associated with better long-term response (P<0.01). Adverse events were generally mild-moderate, but 3 patients developed serum sickness and 2 colitis; there were no sequelae. Dexamethasone could be difficult to tolerate. Fourteen patients became hypogammaglobulinemic and half had increased frequency of minor infections; 9 of 12 evaluable patients recovered their IgG levels. Rituximab combined with three cycles of dexamethasone provides apparently better results to reported findings with rituximab alone, dexamethasone alone, or the combination with one cycle of dexamethasone. The results suggest medical cure may be achievable in immunothrombocytopenia, especially in women and in patients within two years of diagnosis. (clinicaltrials.gov identifier:02050581) Rituximab and three dexamethasone cycles provide responses similar to splenectomy in women and those with immune thrombocytopenia of less than two years duration