Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress

The emergence of new primate-specific genes is an essential factor in human and primate brain development and functioning. POU2F1/Oct-1 is a transcription regulator in higher eukaryotes which is involved in the regulation of development, differentiation, stress response, and other processes. We have demonstrated that the Tigger2 transposon insertion into the POU2F1 gene which occurred in the primate lineage led to the formation of an additional exon (designated the Z-exon). Z-exon-containing primate-specific Oct-1Z transcript includes a short upstream ORF (uORF) located at its 5’-end and the main ORF encoding the Oct-1Z protein isoform (Pou2F1 isoform 3, P14859-3), which differs from other Oct-1 isoforms by its N-terminal peptide. The Oct-1Z-encoding transcript is expressed mainly in human brain cortex. Under normal conditions, the translation of the ORF coding for the Oct-1Z isoform is repressed by uORF. Under various stress conditions, uORF enables a strong increase in the translation of the Oct-1Z-encoding ORF. Increased Oct-1Z expression levels in differentiating human neuroblasts activate genes controlling stress response, neural cell differentiation, brain formation, and organogenesis. We have shown that the Oct-1Z isoform of the POU2F1/Oct-1 transcription factor is an example of a primate-specific genomic element contributing to brain development and cellular stress defense.

Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5′-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.

Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

A novel isoform of cryptochrome 4 (Cry4b) is expressed in the retina of a night-migratory songbird

The primary sensory molecule underlying light-dependent magnetic compass orientation in migratory birds has still not been identified. The cryptochromes are the only known class of vertebrate proteins which could mediate this mechanism in the avian retina. Cryptochrome 4 of the night-migratory songbird the European robin (Erithacus rubecula; erCry4) has several of the properties needed to be the primary magnetoreceptor in the avian eye. Here, we report on the identification of a novel isoform of erCry4, which we named erCry4b. Cry4b includes an additional exon of 29 amino acids compared to the previously described form of Cry4, now called Cry4a. When comparing the retinal circadian mRNA expression pattern of the already known isoform erCry4a and the novel erCry4b isoform, we find that erCry4a is stably expressed throughout day and night, whereas erCry4b shows a diurnal mRNA oscillation. The differential characteristics of the two erCry4 isoforms regarding their 24-h rhythmicity in mRNA expression leads us to suggest that they might have different functions. Based on the 24-h expression pattern, erCry4a remains the more likely cryptochrome to be involved in radical-pair-based magnetoreception, but at the present time, an involvement of erCry4b cannot be excluded.

Transcription-coupled repair in Drosophila melanogaster is independent of the mismatch repair pathway

Transcription-coupled repair (TCR) removes base damage on the transcribed strand of a gene to ensure a quick resumption of transcription. Based on the absence of key enzymes for TCR and empirical evidence, TCR was thought to be missing in Drosophila melanogaster. The recent demonstration of TCR in S2 cells raises the question about the involved genes. Since the mismatch repair (MMR) pathway serves a central role in TCR, at least in Escherichia coli, we studied the mutational signatures in flies with a deletion of the MMR gene spellchecker1 (spel1), a MutS homolog. Whole-genome sequencing of mutation accumulation (MA) lines obtained 7,345 new single nucleotide variants (SNVs) and 5,672 short indel mutations, the largest data set from an MA study in D. melanogaster. Based on the observed mutational strand-asymmetries, we conclude that TCR is still active without spel1. The operation of TCR is further confirmed by a negative association between mutation rate and gene expression. Surprisingly, the TCR signatures are detected for introns, but not for exons. We propose that an additional exon-specific repair pathway is masking the signature of TCR. This study presents the first step towards understanding the molecular basis of TCR in Drosophila melanogaster.

184 ALTERNATIVE SPLICING OF DNA METHYLTRANSFERASE 1 IN PORCINE OOCYTES

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as of cloned embryos. The objective of this study was to identify and sequence isoforms of Dnmt1 expressed in porcine oocytes. Total RNA was isolated from pools of 50 denuded mature oocytes as well as from fibroblast cells using the Trizol method. RNA was co-precipitated with glycogen, and residual genomic DNA was removed with DNase I. A RACE System (Invitrogen, Carlsbad, CA, USA) was used to amplify the 5′ cDNA end of Dnmt1. Briefly, the first strand of cDNA was synthesized using SuperScript II and a Dnmt1-specific primer followed by degradation of the RNA strands and incorporation of TdT and dCTP tails to the 3′ ends of the cDNA. Tailed cDNA was amplified by PCR using a forward anchor primer and a reverse Dnmt1-specific primer. PCR products were separated by electrophoresis on an agarose gel. Resulting PCR products were subcloned into a cloning vector, and the cDNA inserts were sequenced. PCR primers capable of amplifying all possible alternatively spliced isoforms of Dnmt1 were used to identify the presence of the RNA sequences found in fibroblasts and oocytes of pigs. Two distinctive bands (290 and 390 bp) were observed after 5′RACE, PCR, and electrophoresis of oocyte Dnmt1 cDNA. Only 1 band of 290 bp was observed after amplification of fibroblast cDNA. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa Dnmt1 genomic sequence located on chromosome 2 (CU462940). The smaller 290-bp band, amplified from both oocytes and fibroblasts, had an identical DNA sequence (EU908731). Structure analysis of the larger 390-bp band indicates that this oocyte Dnmt1 isoform has an additional exon located between exon 1 and 2 of the somatic form of Dnmt1 (EU908730). PCR products amplified using primers specific for the oocyte or somatic transcript verified the presence of the additional exon in the oocyte Dnmt1 splicing variant. In conclusion, this study shows that porcine oocytes express an alternative isoform of Dnmt1 in addition to the somatic transcript. The 2 identified isoforms are produced by alternative splicing of the Dnmt1 gene. Analysis of the oocyte and somatic Dnmt1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the “secretory” NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

The Tetratricopeptide Repeat Domain 7 Gene is Mutated in Flaky Skin Mice: A Model for Psoriasis, Autoimmunity, and Anemia

The flaky skin (fsn) mutation in mice causes pleiotropic abnormalities including psoriasiform dermatitis, anemia, hyper-IgE, and anti-dsDNA autoantibodies resembling those detected in systemic lupus erythematosus. The fsn mutation was mapped to an interval of 3.9 kb on chromosome 17 between D17Mit130 and D17Mit162. Resequencing of known and predicted exons and regulatory sequences from this region in fsn/fsn and wild-type mice indicated that the mutation is due to the insertion of an endogenous retrovirus (early transposon class) into intron 14 of the Tetratricopeptide repeat (TPR) domain 7 (Ttc7) gene. The insertion leads to reduced levels of wild-type Ttc7 transcripts in fsn mice and the insertion of an additional exon derived from the retrovirus into the majority of Ttc7 mRNAs. This disrupts one of the TPRs within TTC7 and may affect its interaction with an as-yet unidentified protein partner. The Ttc7 is expressed in multiple types of tissue including skin, kidney, spleen, and thymus, but is most abundant in germinal center B cells and hematopoietic stem cells, suggesting an important role in the development of immune system cells. Its role in immunologic and hematologic disorders should be either investigated.