Protein kinase CK2 sustains de novo fatty acid synthesis by regulating the expression of SCD-1 in human renal cancer cells.

Background: Clear cell renal cell carcinoma (ccRCC) is a type of cancer characterized by a vast intracellular accumulation of lipids that are critical to sustain growth and viability of the cells in the tumour microenvironment. Stearoyl-CoA 9-desaturase 1 (SCD-1) is an essential enzyme for the synthesis of monounsaturated fatty acids and consistently overexpressed in all stages of ccRCC growth.

Methods: Human clear cell renal cell carcinoma lines were treated with small-molecule inhibitors of protein kinase CK2.

Body weight control via protein kinase CK2: diet-induced obesity counteracted by pharmacological targeting.

Background: Protein kinase CK2 is a highly conserved enzyme implicated in the pathogenesis of various human illnesses including obesity. Despite compelling evidence for the involvement of this kinase in the pathophysiology of obesity, the molecular mechanisms by which CK2 might regulate fat metabolism are still poorly understood.

Methods And Results: In this study, we aimed to elucidate the role of CK2 on lipid metabolism by employing both in vitro and in vivo approaches using mouse pre-adipocytes and a mouse model of diet-induced obesity.

Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition.

All exons are not created equal-exon vulnerability determines the effect of exonic mutations on splicing.

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements (SREs), but there is a misconception that all exons have an equal dependency on SREs and thus a similar vulnerability to aberrant splicing. We demonstrate that some exons are more likely to be affected by exonic splicing mutations (ESMs) due to an inherent vulnerability, which is context dependent and influenced by the strength of exon definition. We have developed VulExMap, a tool which is based on empirical data that can designate whether a constitutive exon is vulnerable.

CRISPR activation to characterize splice-altering variants in easily accessible cells.

Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types.

Regulating gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The c.

Trans-Regulation of Alternative mRNA Processing by CDK12 in Non-Small-Cell Lung Cancer Cells.

Immunotherapy using checkpoint inhibitors targeting the interaction between PD-1 on T cells and PD-L1 on cancer cells has shown significant results in non-small-cell lung cancer (NSCLC). Not all patients respond to the therapy, and PD-L1 expression heterogeneity is proposed to be one determinant for this. The alternative processing of RNA, which depends on an alternative poly-A site in intron 4, generates a shorter mRNA variant () encoding soluble PD-L1 (sPD-L1), relative to the canonical mRNA encoding membrane-associated PD-L1 (mPD-L1).

Candidate genes and sequence variants for susceptibility to mycobacterial infection identified by whole-exome sequencing.

Inborn errors of immunity are known to influence susceptibility to mycobacterial infections. The aim of this study was to characterize the genetic profile of nine patients with mycobacterial infections (eight with BCGitis and one with disseminated tuberculosis) from the Republic of Moldova using whole-exome sequencing. In total, 12 variants in eight genes known to be associated with Mendelian Susceptibility to Mycobacterial Disease (MSMD) were detected in six out of nine patients examined.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples.

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene.

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping.

Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling.

-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy.

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity.

Vulnerable exons, like ACADM exon 5, are highly dependent on maintaining a correct balance between splicing enhancers and silencers.

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements, but there is a misconception that all exons have an equal dependency on splicing regulatory elements and thus a similar susceptibility to aberrant splicing. We investigated exonic mutations in ACADM exon 5 to experimentally examine their effect on splicing and found that 7 out of 11 tested mutations affected exon inclusion, demonstrating that this constitutive exon is particularly vulnerable to exonic splicing mutations. Employing ACADM exon 5 and 6 as models, we demonstrate that the balance between splicing enhancers and silencers, flanking intron length, and flanking splice site strength are important factors that determine exon definition and splicing efficiency of the exon in question.

Pseudoexon activation in disease by non-splice site deep intronic sequence variation - wild type pseudoexons constitute high-risk sites in the human genome.

Accuracy of pre-messenger RNA (pre-mRNA) splicing is crucial for normal gene expression. Complex regulation supports the spliceosomal distinction between authentic exons and the many seemingly functional splice sites delimiting pseudoexons. Pseudoexons are nonfunctional intronic sequences that can be activated for aberrant inclusion in mRNA, which may cause disease.

Use of Molecular Genetic Analyses in Danish Routine Newborn Screening.

Historically, the analyses used for newborn screening (NBS) were biochemical, but increasingly, molecular genetic analyses are being introduced in the workflow. We describe the application of molecular genetic analyses in the Danish NBS programme and show that second-tier molecular genetic testing is useful to reduce the false positive rate while simultaneously providing information about the precise molecular genetic variant and thus informing therapeutic strategy and easing providing information to parents. When molecular genetic analyses are applied as second-tier testing, valuable functional data from biochemical methods are available and in our view, such targeted NGS technology should be implemented when possible in the NBS workflow.

Identification of SRSF10 as a regulator of SMN2 ISS-N1.

Understanding the splicing code can be challenging as several splicing factors bind to many splicing-regulatory elements. The SMN1 and SMN2 silencer element ISS-N1 is the target of the antisense oligonucleotide drug, Spinraza, which is the treatment against spinal muscular atrophy. However, limited knowledge about the nature of the splicing factors that bind to ISS-N1 and inhibit splicing exists.

Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27 in Conditions Unfavorable for the Growth of Myoblast Cells.

Background/aims: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated.

Methods: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth.

DeepCLIP: predicting the effect of mutations on protein-RNA binding with deep learning.

Nucleotide variants can cause functional changes by altering protein-RNA binding in various ways that are not easy to predict. This can affect processes such as splicing, nuclear shuttling, and stability of the transcript. Therefore, correct modeling of protein-RNA binding is critical when predicting the effects of sequence variations.

Danish expanded newborn screening is a successful preventive public health programme.

Introduction: Newborn screening is a public health programme for early diagnosis of treatable diseases.

Methods: The subjects included were newborns born 2002-2019. Expanded newborn screening (eNBS) for metabolic diseases was introduced as a pilot project from 2002 to 2009, followed by routine screening with informed dissent.

Down-regulation of CK2α correlates with decreased expression levels of DNA replication minichromosome maintenance protein complex (MCM) genes.

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2β). It is implicated in every stage of the cell cycle and in the regulation of various intracellular pathways associated with health and disease states. The catalytic subunits have similar biochemical activity, however, their functions may differ significantly in cells and in vivo.

A pathogenic haplotype, common in Europeans, causes autosomal recessive albinism and uncovers missing heritability in OCA1.

Oculocutaneous albinism (OCA) is a genetically heterogeneous disorder. Six genes are associated with autosomal recessive OCA (TYR, OCA2, TYRP1, SLC45A2, SLC24A5 and LRMDA), and one gene, GPR143, is associated with X-linked ocular albinism (OA). Molecular genetic analysis provides a genetic diagnosis in approximately 60% of individuals with clinical OA/OCA.

Next generation sequencing of RNA reveals novel targets of resveratrol with possible implications for Canavan disease.

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid β-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity.

Proceedings of the fifth international RASopathies symposium: When development and cancer intersect.

This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer.

Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells.

Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene.