Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens.

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Pan-methylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA, -ADMA and -SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.

Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry.

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light-chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells.

To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non-homologous end-joining. We searched for REV7-associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded DNA-binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re-localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA-damaging agents. In a BRCA1-mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases, revealing promise for FAM35A as a therapeutically relevant cancer marker.

Region-specific activation of oskar mRNA translation by inhibition of Bruno-mediated repression.

A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3' UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation-region-specific activation-which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization.

The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription.

The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We found that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we found thatGli1andNanogare co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway.

Large-scale analysis of post-translational modifications in E. coli under glucose-limiting conditions.

BACKGROUND: Post-translational modification (PTM) of proteins is central to many cellular processes across all domains of life, but despite decades of study and a wealth of genomic and proteomic data the biological function of many PTMs remains unknown. This is especially true for prokaryotic PTM systems, many of which have only recently been recognized and studied in depth. It is increasingly apparent that a deep sampling of abundance across a wide range of environmental stresses, growth conditions, and PTM types, rather than simply cataloging targets for a handful of modifications, is critical to understanding the complex pathways that govern PTM deposition and downstream effects. RESULTS: We utilized a deeply-sampled dataset of MS/MS proteomic analysis covering 9 timepoints spanning the Escherichia coli growth cycle and an unbiased PTM search strategy to construct a temporal map of abundance for all PTMs within a 400 Da window of mass shifts. Using this map, we are able to identify novel targets and temporal patterns for N-terminal N α acetylation, C-terminal glutamylation, and asparagine deamidation. Furthermore, we identify a possible relationship between N-terminal N α acetylation and regulation of protein degradation in stationary phase, pointing to a previously unrecognized biological function for this poorly-understood PTM. CONCLUSIONS: Unbiased detection of PTM in MS/MS proteomics data facilitates the discovery of novel modification types and previously unobserved dynamic changes in modification across growth timepoints.

Useful Web Resources.

An increasing number of web resources are available for aiding in proteomics research. Databases contain repositories of proteins and associated information. A recent article by Chen et al. (Genomics Proteomics Bioinformatics 13(1):36-39, 2015) evaluates a number of MS-based proteomics repositories containing MS and expression data, including repositories devoted to cataloguing high confidence post-translational modifications. Many sites have tools developed by research labs that are shared with the community and online tutorials and videos for learning how to use the tools. This chapter contains a selection of web sites useful for proteomics analyses but is by no means comprehensive. Using a search engine such as Google is the easiest way to find the sites using the name given below.

Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS.

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.

The NFIB/CARM1 partnership is a driver in preclinical models of small cell lung cancer.

The coactivator associated arginine methyltransferase (CARM1) promotes transcription, as its name implies. It does so by modifying histones and chromatin bound proteins. We identified nuclear factor I B (NFIB) as a CARM1 substrate and show that this transcription factor utilizes CARM1 as a coactivator. Biochemical studies reveal that tripartite motif 29 (TRIM29) is an effector molecule for methylated NFIB. Importantly, NFIB harbors both oncogenic and metastatic activities, and is often overexpressed in small cell lung cancer (SCLC). Here, we explore the possibility that CARM1 methylation of NFIB is important for its transforming activity. Using a SCLC mouse model, we show that both CARM1 and the CARM1 methylation site on NFIB are critical for the rapid onset of SCLC. Furthermore, CARM1 and methylated NFIB are responsible for maintaining similar open chromatin states in tumors. Together, these findings suggest that CARM1 might be a therapeutic target for SCLC.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

Phosphorylation by CK2 enhances the rapid light-induced degradation of phytochrome interacting factor 1 in Arabidopsis.

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four ß subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

Tyrosyl-DNA phosphodiesterase 1 excises the 3'-DNA-ALKBH1 cross-link and its application for 3'-DNA-ALKBH1 cross-link characterization by LC-MS/MS.

The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3'-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3' position of the incised AP site, 3'-phospho-α,ß-unsaturated aldehyde (3'-PUA), to form a 3'-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3'-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3'-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3'-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3'-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3'-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3'-PUA.

Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase.

In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.

Horizontal-Acquisition of a Promiscuous Peptidoglycan-Recycling Enzyme Enables Aphids To Influence Symbiont Cell Wall Metabolism.

During evolution, enzymes can undergo shifts in preferred substrates or in catalytic activities. An intriguing question is how enzyme function changes following horizontal gene transfer, especially for bacterial genes that have moved to animal genomes. Some insects have acquired genes that encode enzymes for the biosynthesis of bacterial cell wall components and that appear to function to support or control their obligate endosymbiotic bacteria. In aphids, the bacterial endosymbiont Buchnera aphidicola provides essential amino acids for aphid hosts but lacks most genes for remodeling of the bacterial cell wall. The aphid genome has acquired seven genes with putative functions in cell wall metabolism that are primarily expressed in the aphid cells harboring Buchnera. In analyses of aphid homogenates, we detected peptidoglycan (PGN) muropeptides indicative of the reactions of PGN hydrolases encoded by horizontally acquired aphid genes but not by Buchnera genes. We produced one such host enzyme, ApLdcA, and characterized its activity with both cell wall derived and synthetic PGN. Both ApLdcA and the homologous enzyme in Escherichia coli, which functions as an l,d-carboxypeptidase in the cytoplasmic PGN recycling pathway, exhibit turnover of PGN substrates containing stem pentapeptides and cross-linkages via l,d-endopeptidase activity, consistent with a potential role in cell wall remodeling. Our results suggest that ApLdcA derives its functions from the promiscuous activities of an ancestral LdcA enzyme, whose acquisition by the aphid genome may have enabled hosts to influence Buchnera cell wall metabolism as a means to control symbiont growth and division. IMPORTANCE Most enzymes are capable of performing biologically irrelevant side reactions. During evolution, promiscuous enzyme activities may acquire new biological roles, especially after horizontal gene transfer to new organisms. Pea aphids harbor obligate bacterial symbionts called Buchnera and encode horizontally acquired bacterial genes with putative roles in cell wall metabolism. Though Buchnera lacks cell wall endopeptidase genes, we found evidence of endopeptidase activity among peptidoglycan muropeptides purified from aphids. We characterized a multifunctional, aphid-encoded enzyme, ApLdcA, which displays l,d-endopeptidase activities considered promiscuous for the Escherichia coli homolog, for which these activities do not contribute to its native role in peptidoglycan recycling. These results exemplify the roles of enzyme promiscuity and horizontal gene transfer in enzyme evolution and demonstrate how aphids influence symbiont cell wall metabolism.

SPINDOC binds PARP1 to facilitate PARylation.

SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components.

CK2 phosphorylates a wide variety of substrates, including translation initiation factors. A mass spectrometric approach was used to identify residues phosphorylated by CK2, which may regulate the activity of initiation factors during the translation initiation process in plants. CK2 in vitro phosphorylation sites were identified in wheat and Arabidopsis thaliana eIF2alpha, eIF2beta, eIF5, and wheat eIF3c. Native wheat eIF5 and eIF2alpha were found to have phosphorylation sites that corresponded to some of the in vitro CK2 phosphorylation sites. A large number of the CK2 sites identified in this study are in conserved binding domains that have been implicated in the yeast multifactor complex (eIF1-eIF3-eIF5-eIF2-GTP-Met-tRNA(i)(Met)). This is the first study to demonstrate that plant initiation factors are capable of forming a multifactor complex in vitro. In addition, the interaction of factors within these complexes was enhanced both in vitro and in native extracts by phosphorylation of one or more initiation factors by CK2. The importance of CK2 phosphorylation of eIF5 was evaluated by site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites. Removal of CK2 phosphorylation sites from eIF5 inhibits the CK2-mediated increase in eIF5 interaction with eIF1 and eIF3c in pulldown assays and reduces the eIF5-mediated stimulation of translation initiation in vitro. These results suggest a functional role for CK2 phosphorylation in the initiation of plant translation.

Actin R256 Mono-methylation Is a Conserved Post-translational Modification Involved in Transcription.

Nuclear actin has been elusive due to the lack of knowledge about molecular mechanisms. From actin-containing chromatin remodeling complexes, we discovered an arginine mono-methylation mark on an evolutionarily conserved R256 residue of actin (R256me1). Actin R256 mutations in yeast affect nuclear functions and cause diseases in human. Interestingly, we show that an antibody specific for actin R256me1 preferentially stains nuclear actin over cytoplasmic actin in yeast, mouse, and human cells. We also show that actin R256me1 is regulated by protein arginine methyl transferase-5 (PRMT5) in HEK293 cells. A genome-wide survey of actin R256me1 mark provides a landscape for nuclear actin correlated with transcription. Further, gene expression and protein interaction studies uncover extensive correlations between actin R256me1 and active transcription. The discovery of actin R256me1 mark suggests a fundamental mechanism to distinguish nuclear actin from cytoplasmic actin through post-translational modification (PTM) and potentially implicates an actin PTM mark in transcription and human diseases.