Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements.

There are ~650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease.

Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function.

Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects.

Intergenic Alu exonisation facilitates the evolution of tissue-specific transcript ends.

The 3' untranslated regions (3' UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3' UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3' UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3' UTRs.

iCLIP: protein-RNA interactions at nucleotide resolution.

RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

Clinical and electrophysiology findings in Slovene patients with Leber hereditary optic neuropathy.

BACKGROUND: To report clinical and electrophysiology findings in Slovene patients with Leber hereditary optic neuropathy (LHON). METHODS: Eight patients with LHON (11-26 years; one female and seven males) were examined in acute stages and at follow-up visits by means of Snellen visual acuity, Ishihara color vision, Goldmann or Octopus G2TOP perimetry, fluorescein angiography (FAG), pattern electroretinogram (PERG), visual evoked potentials (VEP) and genetic testing. RESULTS: Patients presented with typical LHON phenotype with bilateral visual acuity loss, abnormal color vision, central scotoma and hyperemic discs with no leakage on FAG. In the acute stage, electrophysiology was performed in 7/8 patients. The PERG P50 component was normal in 14/14 eyes, while the N95 component was reduced in 7/14 eyes. VEP wave P100 was reduced and delayed in 14/14 eyes. In this stage, temporal pallor of the optic disc was visible in 4/7 eyes with reduced PERG N95. At follow-up (1-11 months after), a reduced PERG N95 component was seen in 13/14 eyes and severely affected VEP in all eyes. In the only eye with a normal PERG N95, hyperemic optic disc was seen 5 months after visual acuity loss, while it was atrophic in all the others. Known mutations (14484T>C, 3460G>A) were found in 2/8 patients, while in others high-throughput sequencing identified new potentially pathogenic mutations. CONCLUSIONS: In Leber hereditary optic neuropathy, a reduced N95 component of PERG and severely reduced VEP P100 may be present already in the acute stage of disease, before optic disc pallor appears, suggesting primary dysfunction of retinal ganglion cells.

Identifying Novel Glioma-Associated Noncoding RNAs by Their Expression Profiles.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play a significant role in cancer development as regulators of protein-coding genes. Their dysregulation was in some extent already associated with glioma, the most aggressive primary brain tumours in adults. The correct diagnosis and treatment selection due to high tumour heterogeneity might be difficult and inadequate, resulting in poor prognosis. Studies of expression patterns of noncoding RNAs (ncRNAs) could provide useful insight in glioma molecular development. We used the qPCR approach to screen and investigate the expression of lncRNAs that were previously deregulated in other cancer types. The study showed altered expression levels for numerous lncRNAs across histologically different glioma samples. Validation of few lncRNAs showed association of expression levels with histological subtype and/or malignancy grade. We also observed deregulated and subtype-distinctive expression for four lncRNA-associated miRNAs. Expression of few lncRNAs and miRNA was also associated with patients' survival, showing potential prognostic value. Several ncRNAs, some already related to glioma and some, to the best of our knowledge, investigated for the first time, might be of greater importance in glioma molecular development and progression. Finding the subtype-specific lncRNA and/or miRNA expression patterns may contribute additional information for a more objective classification.

Therapeutic activity of modified U1 core spliceosomal particles.

Modified U1 snRNAs bound to intronic sequences downstream of the 5' splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations.

BBC3 is down-regulated with increased tumor size independently of p53 expression in head and neck cancer.

The study focuses on BBC3 gene expression in primary head and neck squamous cell carcinomas (HNSCC) in relation to pTNM classification. We used quantitative real-time PCR on 35 biopsy samples of HNSCC tumor samples to evaluate differences of BBC3 expression depending on tumor size, regional lymph node involvement, location and tumor staging. In order to confirm the model of BBC3-mediated apoptosis, we used immunohistochemistry to determine the expression of apoptotic proteins p53, p63, Bcl-2 and Bax. We also used publicly available cDNA microarray datasets. Our results show a general down-regulation of BBC3 in tumor tissue compared to adjacent normal tissue. The expression was significantly altered among different groups of patho-histologically evaluated tumor sizes, but not in relation to tumor location, regional lymph node involvement or tumor stage. That suggests the potential use of BBC3 as a new tumor size marker in HNSCC. Through protein expression analysis combined with publicly available cDNA microarray datasets of apoptotic factors, we confirmed the model of BBC3-mediated apoptosis, which can be activated with or without p53.