Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway

The acidic protein rich in leucines Anp32b is an immunomodulator of inflammation in mice

ANP32B belongs to a family of evolutionary conserved acidic nuclear phosphoproteins (ANP32A-H). Family members have been described as multifunctional regulatory proteins and proto-oncogenic factors affecting embryonic development, cell proliferation, apoptosis, and gene expression at various levels. Involvement of ANP32B in multiple processes of cellular life is reflected by the previous finding that systemic gene knockout (KO) of Anp32b leads to embryonic lethality in mice. Here, we demonstrate that a conditional KO of Anp32b is well tolerated in adult animals. However, after immune activation splenocytes isolated from Anp32b KO mice showed a strong commitment towards Th17 immune responses. Therefore, we further analyzed the respective animals in vivo using an experimental autoimmune encephalomyelitis (EAE) model. Interestingly, an exacerbated clinical score was observed in the Anp32b KO mice. This was accompanied by the finding that animal-derived T lymphocytes were in a more activated state, and RNA sequencing analyses revealed hyperactivation of several T lymphocyte-associated immune modulatory pathways, attended by significant upregulation of Tfh cell numbers that altogether might explain the observed strong autoreactive processes. Therefore, Anp32b appears to fulfill a role in regulating adequate adaptive immune responses and, hence, may be involved in dysregulation of pathways leading to autoimmune disorders and/or immune deficiencies

CRM1-mediated nuclear export of CD83 mRNA PRE.

<p><b>A.</b> COS7 cells were transiently transfected either with an expression vector encoding for human CD83 cDNA flanked by the homologous 5′- and 3′-UTR (wt; lane 1–6) or with a related vector in which essential PRE sequences were deleted (PREΔSubL1–3; lane 7–12). At day two posttransfection cultures were exposed to 10 nM of the CRM1 inhibitor LMB or DMSO (solvent control) for 8 hours. Total, cytoplasmic and nuclear RNA was isolated, subjected to CD83- and GAPDH-specific (negative control) PCR and analyzed by gel electrophoresis. <b>B.</b> Quantitative real-time PCR of the RNA probes shown in panel A. RNA ratios +LMB/−LMB are depicted. <b>C.</b> Total, cytoplasmic and nuclear RNA was isolated from cell cultures which were cotransfected with the CD83 PREΔSubL1–3 expression vector and either a construct expressing four tandem repeats of the MPMV CTE (4×CTE) or the respective parental vector (negative control). For NXF1/TAP-independent control, mitochondrial cytochrome C oxidase mRNA was detected in total and cytoplasmic RNA, while GAPDH-specific transcripts were detected in nuclear RNA. <b>D.</b> Quantitative real-time PCR of the RNA probes shown in panel C. RNA ratios +/− NXF1/TAP inactivation (+CTE/−CTE) are depicted.</p

RRM1 and RRM2 of HuR are necessary for efficient CD83 mRNA recognition.

<p><b>A.</b> Schematic diagram of the domain structure of HuR. The positions of the three RNA recognition motifs (RRM1–3) and the hinge region (H) in the human 326 amino acid (aa) HuR protein are indicated. Recombinant proteins (indicated by probe #) which were subsequently analyzed for CD83 PRE binding are depicted. Probe numbers correspond to gel lanes in panel B and C. <b>B.</b> Coomassie-stained SDS-PAGE of the recombinantly expressed and purified proteins (indicated by arrowheads). M, marker proteins. <b>C.</b> Radiolabelled CD83wt PRE coding sequence RNA was incubated either with GST (negative control) or with various HuR-derived GST-fusion proteins. Lane 1: GST; lane 2: full-length HuR; lane 3: HuR aa 1–244 (RRM1-RRM2-H); lane 4: HuR aa 27–93 (RRM1); lane 5: HuR aa 108–174 (RRM2); lane 6: HuR aa 246–317 (RRM3); lane 7: HuR aa 103–244 (RRM2-H); lane 8: HuR aa 190–328 (H-RRM3); HuR aa 175–245 (H). CD83 PRE RNA:protein interaction was analyzed by gel retardation assay as before.</p

HuR binds in a specific manner to the CD83 PRE RNA region.

<p><b>A.</b> Increasing amounts of bacterial expressed GST-HuR protein was incubated either with the radiolabelled CD83 mRNA wildtype (wt) coding sequence (CD83wt; lane 1–7) or the respective probe lacking the HuR target sequence PRE (CD83ΔPRE; lane 8–14). Complex formation was visualized by gel retardation assay and autoradiography. Lane 1: CD83wt RNA alone; lane 2: GST negative control; lane 3–7 increasing amounts of GST-HuR (0.095–0.475 µM); lane 8: CD83ΔPRE alone; lane 9: GST negative control; lane 10–14: increasing amounts of GST-HuR (0.095–0.475 µM). <b>B.</b> Analysis of PRE binding specificity by competition experiments. GST-HuR protein (lane 2–4 and lane 11–13: 0.119 µM, 0.238 µM, 0.475 µM, respectively; lane 5–9 and 14–18: 0.475 µM, respectively) or GST (1 µM) for negative control (lane 1 and 10) was incubated together with radiolabelled CD83wt PRE mRNA and analyzed as before. Increasing amounts (1–5 fold excess over CD83wt PRE mRNA) of either unlabelled HIV-1 RRE RNA (lane 5–9) or unlabelled TNFα ARE RNA (lane 14–18) were added to individual binding reactions.</p

Deletion of individual CD83 PRE substructures impairs binding to HuR.

<p><b>A.</b> GST alone (negative control, 2.2 µM, lane 1, 6, 11, 16) or increasing amounts of recombinant GST-HuR (0.19 µM, 0.38 µM, 0.57 µM, 0.95 µM) were incubated with radiolabelled CD83wt PRE RNA or several combinations of uridine-rich element (URE) mutants as indicated at the top of the panels. Formation of protein-RNA interaction (indicated by arrowhead) was subsequently detected by gel retardation analysis. <b>B.</b> GST alone (negative control; 2.2 µM, lane 1, 5, 9, 13) or increasing amounts of recombinant GST-HuR (0.237 µM, 0.475 µM, 0.95 µM; lanes 2–4, 6–8, 10–12, 14–16, respectively) were incubated together with radiolabelled full-length CD83wt PRE RNA or various PRE subloop (SubL) deletions (depicted at the top of the panels). Complex formation (indicated by arrowhead) was subsequently visualized by gel retardation assay as before. <b>C.</b> COS7 cells were transfected with expression vectors encoding for human CD83 cDNA flanked by the entire homologous 5′- and 3′-UTR (lane 1 and 2) or derivatives thereof (lane 3: ΔSubL1; lane 4: ΔSubL2; lane 5: ΔSubL3). Cellular lysates were subjected to immuno-PCR using anti-HuR antiserum (lane 2–5) or rabbit IgG for negative control (lane 1). CD83-specific RNA was detected by PCR followed by gel electrophoresis. A mock reaction without template served as additional negative control (nc; lane 6). This experiment has been reproduced at least three times with the same results.</p

Major results from the first plasma campaign of the Wendelstein 7-X stellarator

\u3cp\u3eAfter completing the main construction phase of Wendelstein 7-X (W7-X) and successfully commissioning the device, first plasma operation started at the end of 2015. Integral commissioning of plasma start-up and operation using electron cyclotron resonance heating (ECRH) and an extensive set of plasma diagnostics have been completed, allowing initial physics studies during the first operational campaign. Both in helium and hydrogen, plasma breakdown was easily achieved. Gaining experience with plasma vessel conditioning, discharge lengths could be extended gradually. Eventually, discharges lasted up to 6 s, reaching an injected energy of 4 MJ, which is twice the limit originally agreed for the limiter configuration employed during the first operational campaign. At power levels of 4 MW central electron densities reached 3 10\u3csup\u3e19\u3c/sup\u3e m\u3csup\u3e-3\u3c/sup\u3e, central electron temperatures reached values of 7 keV and ion temperatures reached just above 2 keV. Important physics studies during this first operational phase include a first assessment of power balance and energy confinement, ECRH power deposition experiments, 2nd harmonic O-mode ECRH using multi-pass absorption, and current drive experiments using electron cyclotron current drive. As in many plasma discharges the electron temperature exceeds the ion temperature significantly, these plasmas are governed by core electron root confinement showing a strong positive electric field in the plasma centre.\u3c/p\u3

Major results from the first plasma campaign of the Wendelstein 7-X stellarator

After completing the main construction phase of Wendelstein 7-X (W7-X) and successfully commissioning the device, first plasma operation started at the end of 2015. Integral commissioning of plasma start-up and operation using electron cyclotron resonance heating (ECRH) and an extensive set of plasma diagnostics have been completed, allowing initial physics studies during the first operational campaign. Both in helium and hydrogen, plasma breakdown was easily achieved. Gaining experience with plasma vessel conditioning, discharge lengths could be extended gradually. Eventually, discharges lasted up to 6 s, reaching an injected energy of 4 MJ, which is twice the limit originally agreed for the limiter configuration employed during the first operational campaign. At power levels of 4 MW central electron densities reached 3 1019 m-3, central electron temperatures reached values of 7 keV and ion temperatures reached just above 2 keV. Important physics studies during this first operational phase include a first assessment of power balance and energy confinement, ECRH power deposition experiments, 2nd harmonic O-mode ECRH using multi-pass absorption, and current drive experiments using electron cyclotron current drive. As in many plasma discharges the electron temperature exceeds the ion temperature significantly, these plasmas are governed by core electron root confinement showing a strong positive electric field in the plasma centre.Peer reviewe