Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations

Multimodal Nonlinear Microscopy for Therapy Monitoring of Cold Atmospheric Plasma Treatment

Here we report on a non-linear spectroscopic method for visualization of cold atmospheric plasma (CAP)-induced changes in tissue for reaching a new quality level of CAP application in medicine via online monitoring of wound or cancer treatment. A combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence lifetime imaging (2P-FLIM) and second harmonic generation (SHG) microscopy has been used for non-invasive and label-free detection of CAP-induced changes on human skin and mucosa samples. By correlation with histochemical staining, the observed local increase in fluorescence could be assigned to melanin. CARS and SHG prove the integrity of the tissue structure, visualize tissue morphology and composition. The influence of plasma effects by variation of plasma parameters e.g., duration of treatment, gas composition and plasma source has been evaluated. Overall quantitative spectroscopic markers could be identified for a direct monitoring of CAP-treated tissue areas, which is very important for translating CAPs into clinical routine

MicroRNAs miR-19, miR-340, miR-374 and miR-542 regulate MID1 protein expression

<div><p>The MID1 ubiquitin ligase activates mTOR signaling and regulates mRNA translation. Misregulation of MID1 expression is associated with various diseases including midline malformation syndromes, cancer and neurodegenerative diseases. While this indicates that MID1 expression must be tightly regulated to prevent disease states specific mechanisms involved have not been identified. We examined miRNAs to determine mechanisms that regulate MID1 expression. MicroRNAs (miRNA) are small non-coding RNAs that recognize specific sequences in their target mRNAs. Upon binding, miRNAs typically downregulate expression of these targets. Here, we identified four miRNAs, miR-19, miR-340, miR-374 and miR-542 that bind to the 3’-UTR of the MID1 mRNA. These miRNAs not only regulate MID1 expression but also mTOR signaling and translation of disease associated mRNAs and could therefore serve as potential drugs for future therapy development.</p></div

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of AR protein.

<p>SH-SY5Y were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or MID1-specific siRNAs as positive control or a non-specific control siRNA as negative control. Upper panel: Left: MID1 as well as AR protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same membranes as loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p

Model showing the role of MID1 in diverse diseases.

<p>We identified miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p as regulators of MID1. By controlling the expression levels of MID1 these miRNAs may affect several MID1-dependent processes. Functionally, MID1 acts as E3 ubiquitin ligase. Known targets of MID1’s ubiquitin ligase activity include PP2A and Fu. By catalyzing the ubiquitination PP2A MID1 induces the proteasomal degradation of PP2A, thereby reducing PP2A activity towards its target proteins. These include proteins involved in transcription regulation of inflammatory genes via NF-κB signaling, which are essentially involved in chronic inflammatory diseases such as asthma or eosinophilic oesophagitis, as well as the Tau protein, that is important in Alzheimer’s disease. Besides inhibiting PP2A MID1 stimulates the activity of mTOR. PP2A and mTOR regulate phosphorylation and thereby activity of the translational regulator S6K. Via PP2A and mTOR, MID1 controls translation of its target mRNAs. These include APP and BACE1, which play an important role in Alzheimer’s disease, mutant HTT, which causes Huntington’s disease, as we as that androgen receptor (AR), which is involved in prostate cancer. The second known target of MID1’s ubiquitin ligase activity is the kinase Fu. Upon MID1-dependent ubiquitination, this protein gets cleaved, which produces an active truncated protein that regulates the transcription factor GLI3.</p

MiRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-216a-5p, hsa-miR-19b-3p, and hsa-miR-340-5p target the MID1 3’-UTR.

<p>(A) HEK293T cells were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-216a-5p, hsa-miR-19b-3p, and hsa-miR-340-5p), a pool of miRNA inhibitors (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-216a-5p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. 24 hours later the same cells were transfected with a reporter construct carrying renilla luciferase fused to the MID1 3’-UTR sequence (bp 1–1352) as well as firefly luciferase, which is used for normalization. Relative light units of renilla normalized to firefly luciferase are shown. Columns represent mean values +/- SEM (* p < 0.001, n = 13). (B-F) Cotransfection of HEK293T cells with miRNA mimics in combination with reporter constructs carrying renilla luciferase fused to the MID1 3’-UTR sequence (bp 1–1352), in which the seed regions of the specific miRNAs have been mutated (MUT). As in (A) firefly luciferase was used for normalization. The resulting data were normalized to the mean value for a control using the non-mutant (WT) MID1 reporter construct described in (A). Columns represent mean values +/- SEM (* p < 0.01, n = 18).</p

Micro RNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p target endogenous MID1.

<p>(A) HEK293T cells were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. Relative MID1 mRNA expression was analyzed in a qPCR using MID1 specific primers and GAPDH-specific primers. Columns represent mean values +/- SEM (* p < 0.0001, n = 12). (B) HEK293T cells were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. Left: MID1 protein levels were analyzed on a western blot using MID1 specific antibodies (upper blot) or Actin-specific antibodies (lower blot). A representative blot of n = 6 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05). (C) HEK293T cells were transfected with a pool of miRNA inhibitors (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. Left: MID1 protein levels were analyzed on a western blot using MID1 specific antibodies (upper blot) or Actin-specific antibodies (lower blot). A representative blot of n = 6 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of HTT protein.

<p>HEK293T cells stably expressing HTT-exon 1 with 51 CAG-repeats were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or a non-specific control siRNA. Upper panel: Left: HTT-exon 1 protein levels were analyzed on a western blot using HTT specific antibodies or Actin-specific antibodies. A representative blot of n = 7 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05). Lower panel: phospho-S6 (pS6) as well as total S6 protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same blots as a loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p

Targeting of endogenous MID1 by miRNAs hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p leads to a reduction of AR protein.

<p>SH-SY5Y were transfected with a pool of miRNA mimics (hsa-miR-374a-5p, hsa-miR-542-3p, hsa-miR-19b-3p, and hsa-miR-340-5p), or MID1-specific siRNAs as positive control or a non-specific control siRNA as negative control. Upper panel: Left: MID1 as well as AR protein levels were analyzed on a western blot using specific antibodies. Actin was detected on the same membranes as loading control. A representative blot of n = 3 is shown. Right: quantification of blots. Columns represent mean values +/- SEM (* p < 0.05).</p

Regulation of the MID1 protein function is fine-tuned by a complex pattern of alternative splicing

Clinical features of Opitz BBB/G syndrome are confined to defects of the developing ventral midline, whereas the causative gene, MID1, is ubiquitously expressed. Therefore, a non-redundant physiological function of the MID1 product appears to be developmentally restricted. Here, we report the identification of several alternative MID1 exons in human, mouse and fugu. We show that splice variants of the MID1 gene that are comparable in terms of function occur in the three organisms, suggesting an important role in the regulation of the MID1 protein function. Accordingly, we observed differential MID1 transcript patterns in a tissue-specific manner by Northern blot and RT-PCR. The identified splice variants cause loss-of-function effects via several mechanisms. Some introduce a stop codon followed by a novel poly(A+) tail, leading to the formation of C-terminally truncated proteins. Dominant negative effects through altered binding to the MID1-interacting protein agr4 in vitro could be demonstrated in a couple of cases. Others carry premature termination codons without poly(A+) tails. These are degraded by nonsense mediated mRNA decay (NMD). Our data reveal a mechanism conserved in human, mouse and fugu that regulates developmentally restricted MID1 activity and suggest NMD to be critical in the translational regulation of a ubiquitously transcribed mRNA