CELF Family Proteins in Cancer: Highlights on the RNA-Binding Protein/Noncoding RNA Regulatory Axis.

Post-transcriptional modifications to coding and non-coding RNAs are unquestionably a pivotal way in which human mRNA and protein diversity can influence the different phases of a transcript's life cycle. CELF (CUGBP Elav-like family) proteins are RBPs (RNA-binding proteins) with pleiotropic capabilities in RNA processing. Their responsibilities extend from alternative splicing and transcript editing in the nucleus to mRNA stability, and translation into the cytoplasm. In this way, CELF family members have been connected to global alterations in cancer proliferation and invasion, leading to their identification as potential tumor suppressors or even oncogenes. Notably, genetic variants, alternative splicing, phosphorylation, acetylation, subcellular distribution, competition with other RBPs, and ultimately lncRNAs, miRNAs, and circRNAs all impact CELF regulation. Discoveries have emerged about the control of CELF functions, particularly via noncoding RNAs, and CELF proteins have been identified as competing, antagonizing, and regulating agents of noncoding RNA biogenesis. On the other hand, CELFs are an intriguing example through which to broaden our understanding of the RBP/noncoding RNA regulatory axis. Balancing these complex pathways in cancer is undeniably pivotal and deserves further research. This review outlines some mechanisms of CELF protein regulation and their functional consequences in cancer physiology.

Combining NMR Spectroscopy and Molecular Dynamic Simulations to Solve and Analyze the Structure of Protein-RNA Complexes.

Understanding the RNA binding specificity of protein is of primary interest to decipher their function in the cell. Here, we review the methodology used to solve the structures of protein-RNA complexes using solution-state NMR spectroscopy: from sample preparation to structure calculation procedures. We also describe how molecular dynamics simulations can help providing additional information on the role of key amino acid side chains and of water molecules in protein-RNA recognition.

Aromatic side-chain conformational switch on the surface of the RNA Recognition Motif enables RNA discrimination.

The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3' untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5'-UUUAA-3' originating from the COX-2 3'-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets.The RNA Recognition Motif (RRM) is the most ubiquitous RNA binding domain. Here the authors combined NMR and molecular dynamics simulations and show that the RRM RNA binding surface exists in different states and that a conformational switch of aromatic side-chains fine-tunes sequence specific binding affinities.

Downregulation of miR-95-3p inhibits proliferation, and invasion promoting apoptosis of glioma cells by targeting CELF2.

Gliomas are the most common and aggressive types of tumors in human brain, of which the prognosis remains dismal because of their biological behavior. The involvement of miRNAs in tumorigenesis of various kinds of cancers drives us to explore new miRNAs related to gliomas. We measured expression level of miR­95­3p by qRT-PCR in human glioma and non-neoplasm brain tissues and found that higher level of miR­95­3p in glioma tissues of higher grade. Biological functions of miR­95­3p on glioma cells were investigated by MTT assay, flow cytometry and transwell assay. We discovered the cell lines transfected with miR­95­3p ASO (antisense oligonucleotide) had retarded proliferation and invasion but enhanced apoptosis ability. We searched on-line tool Targetscan and selected CELF (CUGBP- and ETR-3-like family 2) as a putative target. Luciferase reporter was employed to confirm the binding sites in 3'UTR region of CELF2 for miR­95­3p. The correlation between expression of CELF2 and miR­95­3p was determined by western blotting and qRT-PCR both in cell lines and human samples. Results showed CELF2 was a direct target of miR­95­3p and expression levels of CELF2 and miR­95­3p were negatively correlated. Finally, CELF2 largely abrogated the effects of miR­95­3p on proliferation, invasion and apoptosis of glioma cells in rescue experiments, which verified the role of CELF2 in miR­95­3p regulating glioma biological behavior. In conclusion, our data suggest the expression level of miR­95­3p is positively related to glioma grade and downregulation of miR­95­3p affects proliferation, invasion and apoptosis of glioma cells by targeting CELF2. We identified miR­95­3p as a putative therapeutic target and CELF2 as a potential tumor suppressor.