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.
Abstract 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.
RNA in DNA repair Vågbø, Cathrine Broberg; Slupphaug, Geir
DNA repair,
November 2020, 2020-11-00, 20201101, Letnik:
95
Journal Article
Recenzirano
Odprti dostop
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.
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.
Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) ...inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.
Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that removes uracil and oxidised pyrimidines from DNA. We show that SMUG1 interacts with the ...pseudouridine synthase Dyskerin (DKC1) and colocalizes with DKC1 in nucleoli and Cajal bodies. As DKC1 functions in RNA processing, we tested whether SMUG1 excised modified bases in RNA and demonstrated that SMUG1 has activity on single-stranded RNA containing 5-hydroxymethyldeoxyuridine, but not pseudouridine, the nucleoside resulting from isomerization of uridine by DKC1. Moreover, SMUG1 associates with the 47S rRNA precursor processed by DKC1, and depletion of SMUG1 leads to a reduction in the levels of mature rRNA accompanied by an increase in polyadenylated rRNA. Depletion of SMUG1, and, in particular, the combined loss of SMUG1 and DKC1, leads to accumulation of 5-hydroxymethyluridine in rRNA. In conclusion, SMUG1 is a DKC1 interaction partner that contributes to rRNA quality control, partly by regulating 5-hydroxymethyluridine levels.
► The base excision repair enzyme SMUG1 interacts with DKC1 ► SMUG1 has activity on ssRNA substrates ► SMUG1 functions in rRNA quality control ► SMUG1 and DKC1 contribute to the control of 5-hm(Urd) levels in rRNA
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.
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•CMTR1 and CMTR2 are essential for mouse embryonic development•Absence of interferon pathway activation in arrested mutant mouse embryos•Chronic activation of the interferon pathway in Cmtr1 mutant livers•Conditional loss of Cmtr1 in the mouse germline leads to infertility
Mammalian RNA cap-proximal ribose methylations are implicated in preventing the activation of the interferon pathway. Dohnalkova et al. reveal that loss of the mouse RNA methylases CMTR1 and CMTR2 causes embryonic developmental arrest without activation of the interferon pathway, pointing to gene regulatory roles.
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.
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.
The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. ...Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.
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