Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.

An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation1,2. Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms3,4. One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Solitary-like and modulated wavepackets in the Couette-Taylor flow with a radial temperature gradient.

We have performed numerical and experimental studies of the flow in a large aspect ratio Couette-Taylor system with a rotating inner cylinder and a fixed radial temperature gradient. The base flow state is a superposition of an azimuthal flow induced by rotation and an axial large convective cell induced by the temperature gradient. For a relatively large temperature gradient, the rotation rate of the inner cylinder destabilizes the convective cell to give rise to travelling wave pattern through a subcritical bifurcation. This wave pattern is associated with a temperature mode and it consists of helical vortices travelling in the annulus. In a small range of the rotation rate, helical vortices have longitudinal meandering leading to the formation of kinks randomly distributed, leading to spatio-temporal disordered patterns. The flow becomes regular for a large interval of rotation rate. The friction, the momentum and the heat transfer coefficients are computed and found to be independent of the heating direction. This article is part of the theme issue 'Taylor-Couette and related flows on the centennial of Taylor's seminal Philosophical Transactions paper (part 1)'.

Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation.

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

Enhancement of Meniscus Pump by Multiple Particles.

We numerically investigate the behavior of a droplet spreading on a smooth substrate with multiple obstacles. As experimental works have indicated, the macroscopic contact line or the three-phase boundary line of a droplet exhibits significant deformation resulting in a local acceleration by successive interactions with an array of tiny obstacles settled on the substrate (Mu et al., Langmuir 2019, 35). We focus on the menisci formation and the resultant pressure and velocity fields inside a liquid film in a two-spherical-particle system to realize an optimal design for the effective liquid-transport phenomenon. Special attention is paid to the meniscus formation around the second particle, which influences the liquid supply related to the pressure difference around the first particle as a function of the distance between the two particles. We find that the meniscus around the first particle plays an additional role as the reservoir of the liquid supplied toward the second particle, which is found to enhance the total pumping effect.

Inwardly Rotating Spirals in a Nonoscillatory Medium.

We report the spontaneous formation of spiral patterns observed at a downward-facing free surface of a horizontal liquid film. The surface is unstable to the Rayleigh-Taylor instability and the resulting liquid discharge from the film can occur in the form of propagating liquid curtains. They are born at the film circular periphery and exhibit patterns of inwardly rotating spiral arms. With the help of a phenomenologically constructed cellular automaton, we show that the patterns arise from the phase locking leading to periodic liquid discharge at constant flow rate over the whole film surface.

Quick Liquid Propagation on a Linear Array of Micropillars.

The wetting process of a high energy surface can be accelerated locally through the capillary interaction of a liquid advancing front with a micro-object introduced to the surface (Mu et al., J. Fluid Mech, 2017, 830, R1). We demonstrate that a linear array of micropillars embedded in a fully wettable substrate can produce quick propagation of liquid along the array. It is observed that multiple interactions of a liquid front with pillars can induce the motion of liquid a hundred times faster than in the absence of pillars.

Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells.

Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E pre-rRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle. Knockdown of PARN or exogenous expression of an enzyme-dead PARN mutant (D28A) accumulated 18S-E in both the cytoplasm and nucleus. Moreover, expression of D28A accumulated 18S-E in Bystin-associated pre-40S particles, suggesting that the enzymatic activity of PARN is necessary for the release of 18S-E from Bystin-associated pre-40S particles. Finally, RNase H-based fragmentation analysis and 3΄-sequence analysis of 18S-E species present in cells expressing wild-type PARN or D28A suggested that PARN degrades the extended regions encompassing nucleotides 5-44 at the 3΄ end of mature 18S rRNA. Our results reveal a novel role for PARN in ribosome biogenesis in human cells.

Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA.

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention.

Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5'-3' exoribonuclease) in the nucleoplasm.

Collaborator of alternative reading frame protein (CARF) associates directly with ARF, p53, and/or human double minute 2 protein (HDM2), a ubiquitin-protein ligase, without cofactors and regulates cell proliferation by forming a negative feedback loop. Although ARF, p53, and HDM2 also participate in the regulation of ribosome biogenesis, the involvement of CARF in this process remains unexplored. In this study, we demonstrate that CARF associates with 5'-3' exoribonuclease 2 (XRN2), which plays a major role in both the maturation of rRNA and the degradation of a variety of discarded pre-rRNA species. We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus. This was also observed upon XRN2 knockdown. Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis. These observations suggest that CARF regulates early steps of pre-rRNA processing during ribosome biogenesis by controlling spatial distribution of XRN2 between the nucleoplasm and nucleolus.

Human nucleolar protein Nop52 (RRP1/NNP-1) is involved in site 2 cleavage in internal transcribed spacer 1 of pre-rRNAs at early stages of ribosome biogenesis.

During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S.

Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis.

The U1 small nuclear ribonucleoprotein (snRNP) plays pivotal roles in pre-mRNA splicing and in regulating mRNA length and isoform expression; however, the mechanism of U1 snRNA quality control remains undetermined. Here, we describe a novel surveillance pathway for U1 snRNP biogenesis. Mass spectrometry-based RNA analysis showed that a small population of SMN complexes contains truncated forms of U1 snRNA (U1-tfs) lacking the Sm-binding site and stem loop 4 but containing a 7-monomethylguanosine 5' cap and a methylated first adenosine base. U1-tfs form a unique SMN complex, are shunted to processing bodies and have a turnover rate faster than that of mature U1 snRNA. U1-tfs are formed partly from the transcripts of U1 genes and partly from those lacking the 3' box elements or having defective SL4 coding regions. We propose that U1 snRNP biogenesis is under strict quality control: U1 transcripts are surveyed at the 3'-terminal region and U1-tfs are diverted from the normal U1 snRNP biogenesis pathway.

Human cell growth regulator Ly-1 antibody reactive homologue accelerates processing of preribosomal RNA.

Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly-1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc-finger DNA-binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre-rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre-rRNAs. In addition, we show that over-expression of LYAR increases cell proliferation without affecting the expression of c-Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.

Splicing factor 2-associated protein p32 participates in ribosome biogenesis by regulating the binding of Nop52 and fibrillarin to preribosome particles.

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.

An allele-selective inter-chromosomal protein bridge supports monogenic antigen expression in the African trypanosome.

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE.

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases. SIGNIFICANCE: Label-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.

Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states.

BACKGROUND: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. RESULTS: Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%. CONCLUSIONS: Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.