TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.

Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894 We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.-Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

A novel family of human leukocyte antigen class II receptors may have its origin in archaic human species.

HLA class II α and ß chains form receptors for antigen presentation to CD4(+) T cells. Numerous pairings of class II α and ß subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αß heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II α and ß heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and ß subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPß sequences, we identified two motifs, Lys-69 and GGPM-(84-87), that are engaged in Ii-dependent assembly of DPß with DRα. We identified five members of a family of DPß chains containing Lys-69 and GGPM 84-87, which assemble with DRα. The Lys/GGPM motif is present in the DPß sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPß mutants with DRα. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.

Stoichiometry of HLA class II-invariant chain oligomers.

BACKGROUND: The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and ß subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αß heterodimers bind to an Ii trimer. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii. CONCLUSION/SIGNIFICANCE: We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii.

Detection of aberrant association of DM with MHC class II subunits in the absence of invariant chain.

Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM alphabeta heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii-/- mice.