Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity [RNA]

May 21st, 2018 by Wei Cui, Ryoma Yoneda, Naomi Ueda, Riki Kurokawa

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 (CCND1) gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS’s nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) in vitro. The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg–Gly–Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike wildtype TLS, an R476A TLS mutant did not inhibit CCND1 promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS–nucleic acid and TLS–protein interactions and thereby participates in transcriptional regulation.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity [RNA]

The ribosome: A hot spot for the identification of new types of protein methyltransferases [Protein Synthesis and Degradation]

May 9th, 2018 by Steven G. Clarke

Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for posttranslationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, while the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins.

Lessons from my undergraduate research students [Computational Biology]

May 9th, 2018 by Paul A Craig

From very early on, my personal/professional life has been shaped by teachers in many different settings. Teaching and learning form a two-way street. In the process of teaching undergraduate students, particularly in the research lab, I have learned some profound lessons about the importance of listening to them, challenging them, giving them autonomy, and allowing them to enjoy success and risk failure. I am now working with a team of faculty members to implement these lessons in a course-based undergraduate research experience in the biochemistry teaching lab. Our goal is to seek answers to the question, "How do students become scientists?" and to implement those answers with our future students.

NMDA receptor-dependent dephosphorylation of serine 387 in Argonaute 2 increases its degradation and affects dendritic spine density and maturation. [Neurobiology]

May 7th, 2018 by Nicolas Paradis-Isler, Jannic Boehm

Argonaute (AGO) proteins are essential components of the microRNA (miRNA) pathway. AGO proteins are loaded with miRNAs to target mRNAs and thereby regulate mRNA stability and protein translation. As such, AGO proteins are important actors in controlling local protein synthesis, for instance, at dendritic spines and synapses. Although miRNA-mediated regulation of dendritic mRNAs has become a focus of intense interest over the past years, the mechanisms regulating neuronal AGO proteins remain largely unknown. Here, using rat hippocampal neurons, we report that dendritic Ago2 is downregulated by the proteasome upon NMDA receptor activation. We found that Ser-387 in Ago2 is dephosphorylated upon NMDA treatment and that this dephosphorylation precedes Ago2 degradation. Expressing Ser-387 phosphorylation-deficient or phosphomimetic Ago2 in neurons, we observed that this phosphorylation site is involved in modulating dendritic spine morphology and postsynaptic density protein 95 (PSD-95) expression in spines. Collectively, our results point toward a signaling pathway linking NMDA receptor-dependent Ago2 dephosphorylation and turnover to postsynaptic structural changes. They support a model in which NMDA receptor-mediated dephosphorylation of Ago2 and Ago2 turnover contribute to the de-repression of mRNAs involved in spine growth and maturation.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on NMDA receptor-dependent dephosphorylation of serine 387 in Argonaute 2 increases its degradation and affects dendritic spine density and maturation. [Neurobiology]

A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila [RNA]

May 7th, 2018 by Rasesh Y. Parikh, Haifan Lin, Vamsi K. Gangaraju

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of anti-sense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells [DNA and Chromosomes]

April 23rd, 2018 by Jun Wu, Pengcheng Wang, Lin Li, Changjun You, Yinsheng Wang

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrated the roles of TLS polymerases in replicative bypass of these lesions in human cells.

Effects of hypo O-GlcNAcylation on Drosophila development [Developmental Biology]

March 27th, 2018 by Daniel N Mariappa, Andrew Ferenbach, Daan M.F. van Aalten

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the polycomb gene O-GlcNAc transferase (OGT, also known as super sex combs [sxc]) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxcK872M, was recessive lethal, whereas a second mutant, the hypomorphic sxcH537A, was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxcH537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxcH537A and a null allele of Drosophila host cell factor (dHcf), encoding an extensively O-GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxcH537A flies lacking a copy of skuld (skd), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxcH537A enabled us to identify pleiotropic effects of globally reduced protein O-GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development.

Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef [Microbiology]

March 27th, 2018 by Amanda J. Chase, Rebecka Wombacher, Oliver T. Fackler

The HIV-1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network (TGN), while the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18 amino acid SH4 domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for TGN retargeting by Nef, as well as the importance of the amino acid at position 8 for determining Nef-sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone (LPC) Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that (i) analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and (ii) intrinsic properties define SH4 domains as cargo of this Nef-sensitive LPC-GTPase transport cycle.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef [Microbiology]

Phospholipid Subcellular Localization and Dynamics [Membrane Biology]

March 27th, 2018 by Yanbo Yang, Minhyoung Lee, Gregory D. Fairn

Membrane biology seeks to understand how lipids and proteins within bilayers assemble into large structures such as organelles and the plasma membrane. Historically, lipids were thought to merely provide structural support for bilayer formation and membrane protein function. Research has now revealed that phospholipid metabolism regulates nearly all cellular processes. Sophisticated techniques helped identify >10,000 lipid species, suggesting that lipids support many biological processes. Here, we highlight the synthesis of the most abundant glycerophospholipids classes and their distribution in organelles. We review vesicular and nonvesicular transport pathways shuttling lipids between organelles and discuss lipid regulators of membrane trafficking and second messengers in eukaryotic cells.

Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by {alpha}-amanitin [Protein Structure and Folding]

March 17th, 2018 by Xiangyang Liu, Lucas Farnung, Christoph Wigge, Patrick Cramer

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II, but forms additional contacts with metazoan-specific residues, which explain why its affinity to mammalian Pol II is ~3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.