Nonsense-Mediated mRNA Decay, a Finely Regulated Mechanism

Nonsense-mediated mRNA decay (NMD) is both a mechanism for rapidly eliminating mRNAs carrying a premature termination codon and a pathway that regulates many genes. This implies that NMD must be subject to regulation in order to allow, under certain physiological conditions, the expression of genes that are normally repressed by NMD. Therapeutically, it might be interesting to express certain NMD-repressed genes or to allow the synthesis of functional truncated proteins. Developing such approaches will require a good understanding of NMD regulation. This review describes the different levels of this regulation in human cells.

Premature Termination Codon in 5′ Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

The cardiac lncRNA Chantico directly regulates Cxcl1 chemokine transcription

In local gene regulation, long noncoding RNA (lncRNA) loci can function at three distinct, but non-mutually exclusive levels: the RNA molecule itself, the process of its transcription, and/or the underlying DNA element. Yet for cis-acting lncRNA, these distinctions are particularly challenging with present tools. To address this problem, we developed Omegazymes, catalytic nucleic acid enzymes to specifically target lncRNA for degradation without triggering premature termination of their transcription. We use Omegazymes to selectively target one of a highly refined set of lncRNAs in the mouse cardiomyocytes, demonstrating that the Chantico lncRNA molecule directly potentiates the transcription of Cxcl1, a neighboring chemokine gene. We find that the Chantico locus also acts at the DNA-level, as the binding of an essential cardiac transcription factor to the Chantico promoter is necessary for Cxcl1 transcription in mature cardiomyocytes. This Chantico regulation of Cxcl1 impacts cardiomyocyte signaling to immune cells, potentially regulating tissue-residence and inflammation.

Countertransference Madness: Supervision, Trans∗, and the Sexual

Drawing on Ferenczi's “confusion of tongues” paradigm, the author argues that the internalization of the supervisor's superego has the potential not only to expand the supervisee's ego (introjection), but also to repress their idiosyncratic functions and attack their thinking activity (intropression). To illustrate this argument, the author recounts his own supervised treatment of a transgender patient during which the supervisor-supervisee transference lapsed into a sadomasochistic dialectic and a folie à deux, leading to the premature termination of both the therapy and the supervision. While the initial interpretation of this experience underscored the supervisor's transphobia, the après-coup of writing up the case has revealed more complex thinking. Accordingly, the countertransference madness to which the author succumbed with his supervisor can now be understood as the unbinding of repressed infantile sexuality and the reenactment of paradoxical scenarios that the patient experienced with his parents.

A 1-bp deletion in bovine QRICH2 causes low sperm count and immotile sperm with multiple morphological abnormalities

Background: Semen quality and male fertility are monitored in artificial insemination bulls to ensure high insemination success rates. Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced twelve ejaculates with an aberrantly low number of sperm (0.2±0.2 x 109 sperm per ml) which were mostly immotile due to multiple morphological abnormalities. Results: The genome of the bull was sequenced at 12-fold coverage to investigate a suspected genetic cause. Comparing the sequence variant genotypes of the bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence of QRICH2 encoding glutamine rich 2 as a compelling candidate causal variant. The 1-bp deletion causes a frameshift in translation and induces a premature termination codon (ENSBTAP00000018337.1:p.Cys1644AlafsTer52). The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subjected to nonsense-mediated mRNA decay. The 1-bp deletion resides on a 675 kb haplotype spanning 181 SNPs from the Illumina BovineHD Bead chip. The haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. This analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities primarily affecting the sperm flagellum and, to a lesser extent, the sperm head. Conclusions: A recessive loss-of-function allele of bovine QRICH2 likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.

The 15 Minute Case Conceptualization

Once understood as useful but optional, case conceptualization is now considered essential and one of the most important of all clinical skills and competencies. As clinicians look for resources to assist in learning and mastering this competency, they must choose among different case conceptualization approaches. They would do well to give serious consideration to those that are both clinically effective and clinician friendly. A truly clinically effective approach explains and guides treatment, and most importantly, predicts challenges and obstacles that are likely to arise over the course of treatment. Most approaches emphasize the functions of explanation and guiding treatment, but seldom include third function which help anticipates likely challenges, which if not proactively addressed are likely to result in therapy interference or premature termination. This function is essential in determining the course and overall effectiveness of therapy. A truly clinician-friendly approach is one that is quick to use and easy to master, and very few approaches can make this claim. The 15 Minute Case Conceptualization is the only approach that is both highly effective and clinician friendly. Research confirms that such case conceptualizations can be completed in only 15 minutes. It is an evidence-based, step-by-step approach that therapists and other mental health professionals need and want.

Molecular Approaches Fighting Nonsense

Nonsense mutations are the result of single nucleotide substitutions in the DNA that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of the mRNA [...]