Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3′-untranslated region (UTR), but also in the coding region (CR) and 5′-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3′UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT–qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns

Analysis of Nitric Oxide-Stabilized mRNAs in Human Fibroblasts Reveals HuR-Dependent Heme Oxygenase 1 Upregulation▿ †

We previously observed that nitric oxide (NO) exposure increases the stability of mRNAs encoding heme oxygenase 1 (HO-1) and TIEG-1 in human and mouse fibroblasts. Here, we have used microarrays to look broadly for changes in mRNA stability in response to NO treatment. Using human IMR-90 and mouse NIH 3T3 fibroblasts treated with actinomycin D to block de novo transcription, microarray analysis suggested that the stability of the majority of mRNAs was unaffected. Among the mRNAs that were stabilized by NO treatment, seven transcripts were found in both IMR-90 and NIH 3T3 cells (CHIC2, GADD45B, HO-1, PTGS2, RGS2, TIEG, and ID3) and were chosen for further analysis. All seven mRNAs showed at least one hit of a signature motif for the stabilizing RNA-binding protein (RBP) HuR; accordingly, ribonucleoprotein immunoprecipitation analysis revealed that all seven mRNAs associated with HuR. In keeping with a functional role of HuR in the response to NO, a measurable fraction of HuR increased in the cytoplasm following NO treatment. However, among the seven transcripts, only HO-1 mRNA showed a robust increase in the level of its association with HuR following NO treatment. In turn, HO-1 mRNA and protein levels were significantly reduced when HuR levels were silenced in IMR-90 cells, and they were elevated when HuR was overexpressed. In sum, our results indicate that NO stabilizes mRNA subsets in fibroblasts, identify HuR as an RBP implicated in the NO response, reveal that HuR alone is insufficient for stabilizing several mRNAs by NO, and show that HO-1 induction by NO is regulated by HuR

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of a female-specific lipocalin (FLP) expressed in the lacrimal glands of Syrian hamsters

A female-specific lacrimal protein from Syrian hamsters has been crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P212121 and diffraction data were collected to 1.86 Å resolution

HuR recruits let-7/RISC to repress c-Myc expression

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. Here, we show that the RBP HuR reduced c-Myc expression by associating with the c-Myc 3′ untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieved the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism, as let-7 required HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. Our findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3′UTR