Progression of monoclonal gammopathy of undetermined significance to multiple myeloma is associated with enhanced translational quality control and overall loss of surface antigens.

BACKGROUND: Despite significant advancements in treatment strategies, multiple myeloma remains incurable. Additionally, there is a distinct lack of reliable biomarkers that can guide initial treatment decisions and help determine suitable replacement or adjuvant therapies when relapse ensues due to acquired drug resistance. METHODS: To define specific proteins and pathways involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM), we have applied super-SILAC quantitative proteomic analysis to CD138 + plasma cells from 9 individuals with MGUS and 37 with MM. RESULTS: Unsupervised hierarchical clustering defined three groups: MGUS, MM, and MM with an MGUS-like proteome profile (ML) that may represent a group that has recently transformed to MM. Statistical analysis identified 866 differentially expressed proteins between MM and MGUS, and 189 between MM and ML, 177 of which were common between MGUS and ML. Progression from MGUS to MM is accompanied by upregulated EIF2 signaling, DNA repair, and proteins involved in translational quality control, whereas integrin- and actin cytoskeletal signaling and cell surface markers are downregulated. CONCLUSION: Compared to the premalignant plasma cells in MGUS, malignant MM cells apparently have mobilized several pathways that collectively contribute to ensure translational fidelity and to avoid proteotoxic stress, especially in the ER. The overall reduced expression of immunoglobulins and surface antigens contribute to this and may additionally mediate evasion from recognition by the immune apparatus. Our analyses identified a range of novel biomarkers with potential prognostic and therapeutic value, which will undergo further evaluation to determine their clinical significance.

Covalent PARylation of DNA base excision repair proteins regulates DNA demethylation.

The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Essential roles of RNA cap-proximal ribose methylation in mammalian embryonic development and fertility.

Eukaryotic RNA pol II transcripts are capped at the 5' end by the methylated guanosine (m7G) moiety. In higher eukaryotes, CMTR1 and CMTR2 catalyze cap-proximal ribose methylations on the first (cap1) and second (cap2) nucleotides, respectively. These modifications mark RNAs as "self," blocking the activation of the innate immune response pathway. Here, we show that loss of mouse Cmtr1 or Cmtr2 leads to embryonic lethality, with non-overlapping sets of transcripts being misregulated, but without activation of the interferon pathway. In contrast, Cmtr1 mutant adult mouse livers exhibit chronic activation of the interferon pathway, with multiple interferon-stimulated genes being expressed. Conditional deletion of Cmtr1 in the germline leads to infertility, while global translation is unaffected in the Cmtr1 mutant mouse liver and human cells. Thus, mammalian cap1 and cap2 modifications have essential roles in gene regulation beyond their role in helping cellular transcripts to evade the innate immune system.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Insight into ALKBH8-related intellectual developmental disability based on the first pathogenic missense variant.

ALKBH8 is a methyltransferase that modifies tRNAs by methylating the anticodon wobble uridine residue. The syndrome of ALKBH8-related intellectual developmental disability (MRT71) has thus far been reported solely in the context of homozygous truncating variants that cluster in the last exon. This raises interesting questions about the disease mechanism, because these variants are predicted to escape nonsense mediated decay and yet they appear to be loss of function. Furthermore, the limited class of reported variants complicates the future interpretation of missense variants in ALKBH8. Here, we report a consanguineous family in which two children with MRT71-compatible phenotype are homozygous for a novel missense variant in the methyltransferase domain. We confirm the pathogenicity of this variant by demonstrating complete absence of ALKBH8-dependent modifications in patient cells. Targeted proteomics analysis of ALKBH8 indicates that the variant does not lead to loss of ALKBH8 protein expression. This report adds to the clinical delineation of MRT71, confirms loss of function of ALKBH8 as the disease mechanism and expands the repertoire of its molecular lesions.

ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA.

BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

Inducible TDG knockout models to study epigenetic regulation.

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

RNA in DNA repair.

Our genome is constantly subject to damage from exogenous and endogenous sources, and cells respond to such damage by initiating a DNA damage response (DDR). Failure to induce an adequate DDR can result in increased mutation load, chromosomal aberrations and a variety of human diseases, including cancer. A rapidly growing body of evidence suggests that a large number of RNA binding proteins are involved in the DDR, and several canonical DNA repair factors have moonlighting functions in RNA metabolism. RNA polymerases and RNA itself have been implicated at various stages of the DDR, including damage sensing, recruitment of DNA repair factors and tethering of broken DNA ends. RNA may even serve as a template for DNA repair under certain conditions. Given the vast number of non-coding RNAs in cells, we have barely started to decipher their potential involvement in genomic maintenance and future research on the interrelationship between RNA and DNA repair may open entirely new treatment options for human disease.

The Mammalian Cap-Specific m6Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues.

The 5' end of eukaryotic mRNAs is protected by the m7G-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, whereas an adenosine at this position is further methylated at the N6 position (m6A) by the mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1) to generate m6Am. Here, we show that although the loss of cap-specific m6Am in mice does not affect viability or fertility, the Pcif1 mutants display reduced body weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific m6Am modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically dead, but like its mammalian counterpart, it retains the ability to associate with the Ser5-phosphorylated CTD of RNA polymerase II (RNA Pol II). Finally, we show that the Trypanosoma Pcif1 is an m6Am methylase that contributes to the N6,N6,2'-O-trimethyladenosine (m62Am) in the hypermethylated cap4 structure of trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.

Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma.

Endonuclease V (EndoV) is a conserved inosine-specific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV-/- tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV-/- livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to fine-tune gene expression. The EndoV-/- tumor suppressive phenotype calls for related studies in human HCC.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication.

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.

Recessive Truncating Mutations in ALKBH8 Cause Intellectual Disability and Severe Impairment of Wobble Uridine Modification.

The wobble hypothesis was proposed to explain the presence of fewer tRNAs than possible codons. The wobble nucleoside position in the anticodon stem-loop undergoes a number of modifications that help maintain the efficiency and fidelity of translation. AlkB homolog 8 (ALKBH8) is an atypical member of the highly conserved AlkB family of dioxygenases and is involved in the formation of mcm5s2U, (S)-mchm5U, (R)-mchm5U, mcm5U, and mcm5Um at the anticodon wobble uridines of specific tRNAs. In two multiplex consanguineous families, we identified two homozygous truncating ALKBH8 mutations causing intellectual disability. Analysis of tRNA derived from affected individuals showed the complete absence of these modifications, consistent with the presumptive loss of function of the variants. Our results highlight the sensitivity of the brain to impaired wobble modification and expand the list of intellectual-disability syndromes caused by mutations in genes related to tRNA modification.

The DNA modification N6-methyl-2'-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction.

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2'-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.

Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.

Genome-wide profiling of DNA 5-hydroxymethylcytosine during rat Sertoli cell maturation.

Sertoli cells have dual roles during the cells' lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression.

Proteome alterations associated with transformation of multiple myeloma to secondary plasma cell leukemia.

Plasma cell leukemia is a rare and aggressive plasma cell neoplasm that may either originate de novo (primary PCL) or by leukemic transformation of multiple myeloma (MM) to secondary PCL (sPCL). The prognosis of sPCL is very poor, and currently no standard treatment is available due to lack of prospective clinical studies. In an attempt to elucidate factors contributing to transformation, we have performed super-SILAC quantitative proteome profiling of malignant plasma cells collected from the same patient at both the MM and sPCL stages of the disease. 795 proteins were found to be differentially expressed in the MM and sPCL samples. Gene ontology analysis indicated a metabolic shift towards aerobic glycolysis in sPCL as well as marked down-regulation of enzymes involved in glycan synthesis, potentially mediating altered glycosylation of surface receptors. There was no significant change in overall genomic 5-methylcytosine or 5-hydroxymethylcytosine at the two stages, indicating that epigenetic dysregulation was not a major driver of transformation to sPCL. The present study constitutes the first attempt to provide a comprehensive map of the altered protein expression profile accompanying transformation of MM to sPCL in a single patient, identifying several candidate proteins that can be targeted by currently available small molecule drugs. Our dataset furthermore constitutes a reference dataset for further proteomic analysis of sPCL transformation.