Control of mRNA Splicing by Intragenic RNA Activators of Stress Signaling: Potential Implications for Human Disease

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100–200 nucleotides in length within primary transcripts of cellular genes, exemplified by the tumor necrosis factor (TNF)-α gene and fetal and adult globin genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA. The TNF-α RNA activator of PKR folds into a compact pseudoknot that is highly conserved within the phylogeny. Upon excision of β-globin first intron, the RNA activator of PKR, located in exon 1, is silenced through strand displacement by a short sequence within exon 2, restricting thereby the ability to activate PKR to the splicing process without impeding subsequent synthesis of β-globin essential for survival. This activator/silencer mechanism likewise controls splicing of α-globin pre-mRNA, but the exonic locations of PKR activator and silencer sequences are reversed, demonstrating evolutionary flexibility. Impaired splicing efficiency may underlie numerous human β-thalassemia mutations that map to the β-globin RNA activator of PKR or its silencer. Even where such mutations change the encoded amino acid sequence during subsequent translation, they carry the potential of first impairing PKR-dependent mRNA splicing or shutoff of PKR activation needed for optimal translation

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well

Subcortical brain volume, regional cortical thickness, and cortical surface area across disorders: findings from the ENIGMA ADHD, ASD, and OCD Working Groups

Objective Attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and obsessive-compulsive disorder (OCD) are common neurodevelopmental disorders that frequently co-occur. We aimed to directly compare all three disorders. The ENIGMA consortium is ideally positioned to investigate structural brain alterations across these disorders. Methods Structural T1-weighted whole-brain MRI of controls (n=5,827) and patients with ADHD (n=2,271), ASD (n=1,777), and OCD (n=2,323) from 151 cohorts worldwide were analyzed using standardized processing protocols. We examined subcortical volume, cortical thickness and surface area differences within a mega-analytical framework, pooling measures extracted from each cohort. Analyses were performed separately for children, adolescents, and adults using linear mixed-effects models adjusting for age, sex and site (and ICV for subcortical and surface area measures). Results We found no shared alterations among all three disorders, while shared alterations between any two disorders did not survive multiple comparisons correction. Children with ADHD compared to those with OCD had smaller hippocampal volumes, possibly influenced by IQ. Children and adolescents with ADHD also had smaller ICV than controls and those with OCD or ASD. Adults with ASD showed thicker frontal cortices compared to adult controls and other clinical groups. No OCD-specific alterations across different age-groups and surface area alterations among all disorders in childhood and adulthood were observed. Conclusion Our findings suggest robust but subtle alterations across different age-groups among ADHD, ASD, and OCD. ADHD-specific ICV and hippocampal alterations in children and adolescents, and ASD-specific cortical thickness alterations in the frontal cortex in adults support previous work emphasizing neurodevelopmental alterations in these disorders

Mutations at various transcription factors binding sites affect protein complexes formation at the <i>vav1</i> promoter.

<p>Electrophoretic mobility shift assay (EMSA) with Jurkat and H441 nuclear extracts was performed in the presence of lil46-47 digoxigenin-labeled probe (nucleotides −98 to +28 of <i>vav1</i> promoter). To produce the mutant oligonucleotides, the corresponding mutated plasmids (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-g002" target="_blank">Fig. 2B</a> schematic) were used as template for the PCR. A schematic of <i>vav</i>1 5′ regulatory sequences, exon 1 and relative oligonucleotide position is shown at the bottom. Bound protein complexes are numbered 1 to 5. The arrow shows the position of complex 5, the heaviest complex that is sensitive to the mutations introduced into the oligonucleotide sequence. The bottom panels of the figure schematically show the relative intensity of bands 1–5 of the EMSA experiment as determined by densitometry (ImageJ software).</p

Oligonucleotides used in EMSA, introduced mutations are underlined.

<p>Oligonucleotides used in EMSA, introduced mutations are underlined.</p

Methylation status of CpG dinucleotides in <i>vav1</i> promoter in tissues of different histological origin^*.

<p>*Percent of methylation on each CpG site was evaluated by bisulfite sequencing. Position refers to that of the CpG dinucleotide relatively to transcription start site (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-g001" target="_blank">Fig. 1</a>), and N refers to number of sequenced clones.</p

Mutations at the E2F/NF-e/c-Myb binding site affect binding of protein complexes to the <i>vav1</i> promoter <i>in vitro</i>.

<p>(A) Electrophoretic mobility shift assay (EMSA) with Jurkat nuclear extracts was performed in the presence of digoxigenin-labeled probe spanning nucleotides −45 to 0 of <i>vav1</i> promoter and containing E2F/NF-e/c-Myb and TCFα/PU.1/ELF1 binding sites (lil157-158; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>). The competition assay was performed with the labeled oligonucleotide and unlabeled competitor oligonucleotides with point mutations as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a> in molar ratio of 1∶1 and 1∶5. The arrow shows the position of the complex that demonstrates sensitivity to the introduced mutations. (B) EMSA performed with labeled oligonucleotide containing only E2F/NF-e/c-Myb binding site (lil 87-88; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>).</p

Methylation on CpG dinucleotides at putative transcription factor binding sites changes the affinity of protein complexes for the <i>vav</i>1 regulatory region.

<p>(A) EMSA was performed with Jurkat T cell nuclear extracts and lil3-4 labeled oligonucleotide. The probe was created by annealing complementary oligonucleotides lil79 and lil80 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>).-3′). The following unlabeled competitors were added: unmethylated lil79-80 oligonucleotide (C₃C₄); oligo methylated on both CpG methylation sites (^metC₃^metC₄); oligo methylated only on CpG₃ (^metC₃C₄), or only on CpG₄ (C₃^metC₄). Competitor oligonucleotide was added in an amount equal to the labeled oligo (1∶1) or in 5 molar excess (1∶5).</p

Methylation of CpG sites in the <i>vav</i>1 promoter impairs expression of the reporter gene in various cell lines.

<p>(A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p<0.01, unpaired student T test.</p