Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1 , were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3 0 -position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4.

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm5s2U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm5s2U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

SUMMARY A previously unrecognized mechanism by which large ribonucleoprotein (megaRNP) granules exit the nucleus is by budding through the nuclear envelope (NE). This mechanism is akin to the nuclear egress of Herpes-type viruses and is essential for proper synapse development. However, the molecular machinery required to remodel the NE during this process is unknown. Here we identify Torsin, a AAA-ATPase that in humans is linked to dystonia, as a major mediator of primary megaRNP envelopment during NE-budding. In torsin mutants, megaRNPs accumulate within the perinuclear space and the mRNAs contained within fail to reach synaptic sites, preventing normal synaptic protein synthesis, and thus proper synaptic bouton development. These studies begin to establish the cellular machinery underlying the exit of megaRNPs via budding, offer an explanation to the “nuclear blebbing” phenotype found in dystonia models and provide an important link between Torsin and synaptic phenotypes observed in dystonia.