Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins.

The RNA recognition motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with well-defined specificity would provide valuable tools and an exacting test of the current understanding of specificity. We have redesigned the specificity of an RRM using rational methods and demonstrated retargeting of its activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of a microRNA precursor (pre-miR-21) with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of rationally engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications.

Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer.

Rbfox proteins regulate tissue-specific splicing by targeting a conserved GCAUG sequence within pre-mRNAs. We report here that sequence-specific binding of the conserved Rbfox RRM to miRNA precursors containing the same sequence motif in their terminal loops, including miR-20b and miR-107, suppresses their nuclear processing. The structure of the complex between precursor miR-20b and Rbfox RRM shows the molecular basis for recognition, and reveals changes in the stem-loop upon protein binding. In mammalian cells, Rbfox2 downregulates mature miR-20b and miR-107 levels and increases the expression of their downstream targets PTEN and Dicer, respectively, suggesting that Rbfox2 indirectly regulates many more cellular miRNAs. Thus, some of the widespread cellular functions of Rbfox2 protein are attributable to regulation of miRNA biogenesis, and might include the mis-regulation of miR-20b and miR-107 in cancer and neurodegeneration.

Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.

Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

TERRA stability is regulated by RALY and polyadenylation in a telomere-specific manner.

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.

Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

SCN9Aencodes the voltage-gated sodium channel Na(v)1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374). Both of these mutations map to the pore region of the Na(v)1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7.

Microbiology of wound infection after oral cancer surgery.

INTRODUCTION: The aim of present study is to investigate the most common infection pathogen found in the postoperative wounds, following surgical treatment of oral and oropharyngeal cancer, in order to identify the most suitable antibiotic treatment. PATIENTS AND METHODS: We analyzed patients with squamous cell cancer of oral and oropharyngeal region. In patients who developed postoperative wound infection, wound swabs were taken from three different sites: the cannula, wounds on the neck and wounds in the oral cavity. RESULTS: In total 195 patients were included. The postoperative wound infection was detected in 115 patients (59%). In average, the swabs were taken 8 days after the surgery. The similar bacterial species from all three sites were detected in 24 patients (12,3%). In comparison, we found that there was statistically significant difference in the bacteria abundance from all three sites (p=0,031). There were significantly more bacteria in the wounds of the neck than cannula (p=0,007) and in the wounds in the oral cavity than cannula (p=0,002). No statistically significant difference between the wound on the neck and in the oral cavity was found. The most frequently isolated bacterial family was Enterobacteriaceae. Other more commonly isolated bacteria species were Staphylococcus spp. (G+), Pseudomonas aeruginosa (G-), Corynebacteruim spp. (G +) and Acinetobacter baumanii (G-). CONCLUSION: Based on the most commonly isolated groups of pathogens we concluded that probably the best empiric antibiotic treatment of wound infections until antibiogram is completed might be achieved from the group of aminoglycosides or quinolones. Antibiotic therapy should be reviewed if necessary when antibiogram is completed.

The altered transcriptome of pediatric myelodysplastic syndrome revealed by RNA sequencing.

Pediatric myelodysplastic syndrome (PMDS) is a very rare and still poorly characterized disorder. In this work, we identified novel potential targets of PMDS by determining genes with aberrant expression, which can be correlated with PMDS pathogenesis. We identified 291 differentially expressed genes (DEGs) in PMDS patients, comprising genes involved in the regulation of apoptosis and the cell cycle, ribosome biogenesis, inflammation and adaptive immunity. Ten selected DEGs were then validated, confirming the sequencing data. These DEGs will potentially represent new molecular biomarkers and therapeutic targets for PMDS.