Combinatorial mRNA binding by AUF1 and Argonaute 2 controls decay of selected target mRNAs.

The RNA-binding protein AUF1 binds AU-rich elements in 3'-untranslated regions to regulate mRNA degradation and/or translation. Many of these mRNAs are predicted microRNA targets as well. An emerging theme in post-transcriptional control of gene expression is that RNA-binding proteins and microRNAs co-regulate mRNAs. Recent experiments and bioinformatic analyses suggest this type of co-regulation may be widespread across the transcriptome. Here, we identified mRNA targets of AUF1 from a complex pool of cellular mRNAs and examined a subset of these mRNAs to explore the links between RNA binding and mRNA degradation for both AUF1 and Argonaute 2 (AGO2), which is an essential effector of microRNA-induced gene silencing. Depending on the specific mRNA examined, AUF1 and AGO2 binding is proportional/cooperative, reciprocal/competitive or independent. For most mRNAs in which AUF1 affects their decay rates, mRNA degradation requires AGO2. Thus, AUF1 and AGO2 present mRNA-specific allosteric binding relationships for co-regulation of mRNA degradation.

Prevalence of de novo aortic insufficiency during long-term support with left ventricular assist devices.

BACKGROUND: Left ventricular assist devices (LVADs) are increasingly used as long-term therapy for end-stage heart failure patients. We compared the prevalence of aortic insufficiency (AI) after HeartMate II (HMII) vs HeartMate XVE (HMI) support and assessed the role of aortic root diameter and aortic valve opening in the development of AI. METHOD: Pre-operative and post-operative echocardiograms of 93 HMI and 73 HMII patients who received implants at our center between January 2004 and September 2009 were retrospectively reviewed. After excluding patients with prior or concurrent surgical manipulation of the aortic valve, with baseline AI, or without baseline echoes, 67 HMI and 63 HMII patients were studied. AI was deemed significant if mild to moderate or greater. Pathology reports were reviewed for 77 patients who underwent heart transplant. RESULTS: AI developed in 4 of 67 HMI (6.0%) and in 9 of 63 HMII patients (14.3%). The median times to AI development were 48 days for HMI patients and 90 days for HMII patients. For patients who remained on device support at 6 and 12 months, freedom from AI was 94.5% and 88.9% in HMI patients and 83.6% and 75.2% in HMII patients (log rank p = 0.194). Aortic root diameters, as determined by echocardiography for the patients with AI, trended to be larger at baseline (3.43 ± 0.43 vs 3.15 ± 0.40; p = 0.067) and follow-up (3.58 ± 0.54 vs 3.29 ± 0.50; p = 0.130) compared with those who did not have AI. Aortic root circumferences were assessed directly by a pathologist in those patients who underwent transplant and were significantly larger in HMII patients who had developed AI compared with those patients who did not (8.44 ± 0.89 vs 7.36 ± 1.02 cm; p = 0.034). Lastly, AI was more common in patients whose aortic valve did not open (11 of 26 vs 1 of 14; p = 0.03). CONCLUSION: Aortic insufficiency occurs frequently in patients who receive continuous-flow support with a HMII LVAD, and may be associated with aortic root diameter enlargement and aortic valve opening. These findings warrant a more thorough preoperative patient evaluation and additional studies to investigate the factors, that may be associated with AI development.

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells.

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure.

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.