Regulation of calcineurin, a Ca²⁺/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.

There is increasing evidence of mitochondrial dysfunction in autism spectrum disorders (ASD), but the causal relationships are unclear. In an ASD patient whose identical twin was unaffected, we identified a postzygotic mosaic mutation p.Q639* in the TRAP1 gene, which encodes a mitochondrial chaperone of the HSP90 family. Additional screening of 176 unrelated ASD probands revealed an identical TRAP1 variant in a male patient who had inherited it from a healthy mother. Notably, newly generated knock-in Trap1 p.Q641* mice display ASD-related behavioral abnormalities that are more pronounced in males than in females. Accordingly, Trap1 p.Q641* mutation also resulted in sex-specific changes in synaptic plasticity, the number of presynaptic mitochondria, and mitochondrial respiration. Thus, the TRAP1 p.Q639* mutation is the first example of a monogenic ASD caused by impaired mitochondrial protein homeostasis.

One of the posttranscriptional mechanisms regulating the stability of RNA molecules involves the addition of nontemplated nucleotides to their 3′ ends, a process known as RNA tailing. To systematically investigate the physiological consequences of terminal nucleotidyltransferase TENT2 absence on RNA 3′ end modifications in the mouse hippocampus, we developed a newTent2knockout mouse. Electrophysiological measurements revealed increased excitability inTent2KO hippocampal neurons, and behavioral analyses showed decreased anxiety and improved fear extinction in these mice. At the molecular level, we observed changes in miRNAs’ monoadenylation inTent2KO mouse hippocampus, but found no effect of the TENT2 loss on the mRNAs’ total poly(A) tail length, as measured by direct nanopore RNA sequencing. Moreover, differential expression analysis revealed transcripts related to synaptic transmission to be downregulated in the hippocampus ofTent2knockout mice. These changes may explain the observed behavioral and electrophysiological alterations. Our data thus establish a link between TENT2-dependent miRNA tailing and the balance of inhibitory and excitatory neurotransmission.

One of the post-transcriptional mechanisms regulating the stability of RNA molecules involves the addition of non-templated nucleotides to their 3' ends, a process known as RNA tailing. To systematically investigate the physiological consequences of terminal nucleotidyltransferase TENT2 absence on RNA 3' end modifications in the mouse hippocampus we developed a new Tent2 knockout mouse. Electrophysiological measurements revealed increased excitability in Tent2 KO hippocampal neurons, and behavioral analyses showed decreased anxiety and improved fear extinction in these mice. At the molecular level, we observed a significant contribution of TENT2 to the monoadenylation of various classes of miRNAs, but found no effect of the enzyme's loss on the total poly(A) tail length of mRNAs, as measured by Direct Nanopore RNA sequencing. Alterations in the monoadenylation of a large population of microRNAs affected the overall mRNA abundance, particularly transcripts related synaptic transmission, which were downregulated in the hippocampus of Tent2 knockout mice. These changes explain the observed behavioral and electrophysiological alterations. Our data thus establish a link between TENT2-dependent microRNA tailing and the balance of inhibitory and excitatory neurotransmission.