Microtubule-associated protein MAP1LC3C regulates lysosomal exocytosis and induces zinc reprogramming in renal cancer cells.

Microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C or LC3C) is a member of the microtubule-associated family of proteins that are essential in the formation of autophagosomes and lysosomal degradation of cargo. LC3C has tumor-suppressing activity, and its expression is dependent on kidney cancer tumor suppressors, such as von Hippel-Lindau protein and folliculin. Recently, we demonstrated that LC3C autophagy is regulated by noncanonical upstream regulatory complexes and targets for degradation postdivision midbody rings associated with cancer cell stemness. Here, we show that loss of LC3C leads to peripheral positioning of the lysosomes and lysosomal exocytosis (LE). This process is independent of the autophagic activity of LC3C. Analysis of isogenic cells with low and high LE shows substantial transcriptomic reprogramming with altered expression of zinc (Zn)-related genes and activity of polycomb repressor complex 2, accompanied by a robust decrease in intracellular Zn. In addition, metabolomic analysis revealed alterations in amino acid steady-state levels. Cells with augmented LE show increased tumor initiation properties and form aggressive tumors in xenograft models. Immunocytochemistry identified high levels of lysosomal-associated membrane protein 1 on the plasma membrane of cancer cells in human clear cell renal cell carcinoma and reduced levels of Zn, suggesting that LE occurs in clear cell renal cell carcinoma, potentially contributing to the loss of Zn. These data indicate that the reprogramming of lysosomal localization and Zn metabolism with implication for epigenetic remodeling in a subpopulation of tumor-propagating cancer cells is an important aspect of tumor-suppressing activity of LC3C.

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis.

R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.

Tobacco smoking induces metabolic reprogramming of renal cell carcinoma.

BACKGROUNDClear cell renal cell carcinoma (ccRCC) is the most common histologically defined renal cancer. However, it is not a uniform disease and includes several genetic subtypes with different prognoses. ccRCC is also characterized by distinctive metabolic reprogramming. Tobacco smoking (TS) is an established risk factor for ccRCC, with unknown effects on tumor pathobiology.METHODSWe investigated the landscape of ccRCCs and paired normal kidney tissues using integrated transcriptomic, metabolomic, and metallomic approaches in a cohort of white males who were long-term current smokers (LTS) or were never smokers (NS).RESULTSAll 3 Omics domains consistently identified a distinct metabolic subtype of ccRCCs in LTS, characterized by activation of oxidative phosphorylation (OXPHOS) coupled with reprogramming of the malate-aspartate shuttle and metabolism of aspartate, glutamate, glutamine, and histidine. Cadmium, copper, and inorganic arsenic accumulated in LTS tumors, showing redistribution among intracellular pools, including relocation of copper into the cytochrome c oxidase complex. A gene expression signature based on the LTS metabolic subtype provided prognostic stratification of The Cancer Genome Atlas ccRCC tumors that was independent of genomic alterations.CONCLUSIONThe work identified the TS-related metabolic subtype of ccRCC with vulnerabilities that can be exploited for precision medicine approaches targeting metabolic pathways. The results provided rationale for the development of metabolic biomarkers with diagnostic and prognostic applications using evaluation of OXPHOS status. The metallomic analysis revealed the role of disrupted metal homeostasis in ccRCC, highlighting the importance of studying effects of metals from e-cigarettes and environmental exposures.FUNDINGDepartment of Defense, Veteran Administration, NIH, ACS, and University of Cincinnati Cancer Institute.

Using spectral matching to interpret LC-MS/MS data during RNA modification mapping.

While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).

RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.

Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.

Mass spectrometry of modified RNAs: recent developments.

A common feature of ribonucleic acids (RNAs) is that they can undergo a variety of chemical modifications. As nearly all of these chemical modifications result in an increase in the mass of the canonical nucleoside, mass spectrometry has long been a powerful approach for identifying and characterizing modified RNAs. Over the past several years, significant advances have been made in method development and software for interpreting tandem mass spectra resulting in approaches that can yield qualitative and quantitative information on RNA modifications, often at the level of sequence specificity. We discuss these advances along with instrumentation developments that have increased our ability to extract such information from relatively complex biological samples. With the increasing interest in how these modifications impact the epitranscriptome, mass spectrometry will continue to play an important role in bioanalytical investigations revolving around RNA.

Essentiality of threonylcarbamoyladenosine (t(6)A), a universal tRNA modification, in bacteria.

Threonylcarbamoyladenosine (t(6)A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t(6)A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t(6)A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA(Ile)-lysidine synthetase. We confirm that t(6)A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t(6)A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t(6)A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t(6)A(-) D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t(6)A in tRNAs. Thus, although t(6)A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

Systematic identification of tRNAome and its dynamics in Lactococcus lactis.

Transfer RNAs (tRNAs) through their abundance and modification pattern significantly influence protein translation. Here, we present a systematic analysis of the tRNAome of Lactococcus lactis. Using the next-generation sequencing approach, we identified 40 tRNAs which carry 16 different post-transcriptional modifications as revealed by mass spectrometry analysis. While small modifications are located in the tRNA body, hypermodified nucleotides are mainly present in the anticodon loop, which through wobbling expand the decoding potential of the tRNAs. Using tRNA-based microarrays, we also determined the dynamics in tRNA abundance upon changes in the growth rate and heterologous protein overexpression stress. With a fourfold increase in the growth rate, the relative abundance of tRNAs cognate to low abundance codons decrease, while the tRNAs cognate to major codons remain mostly unchanged. Significant changes in the tRNA abundances are observed upon protein overexpression stress, which does not correlate with the codon usage of the overexpressed gene but rather reflects the altered expression of housekeeping genes.

Metabolic de-isotoping for improved LC-MS characterization of modified RNAs.

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of (16)O/(18)O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural (13)C and (15) N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in (12)C-enriched/(13)C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the (16)O/(18)O differential labeling approach.

The global identification of tRNA isoacceptors by targeted tandem mass spectrometry.

Transfer ribonucleic acids (tRNA) are a biologically significant class of non-coding ribonucleic acids (ncRNAs) that pose unique analytical challenges for complete characterization. Here we present a robust and simple method for the consistent and accurate identification of individual tRNAs from a pool of total tRNA obtained from cell lysate. Through this method individual isoacceptor tRNAs are identified by the detection of unique oligonucleotide sequences which arise from a single enzymatic digestion. These unique sequences can be detected by monitoring specific transitions from precursor to product ions. Thus, for any pool of known tRNA sequences including posttranscriptional modifications, targeted tandem mass spectrometry can be used for monitoring these specific transitions. The proposed method was developed and validated using a set of known tRNAs from Escherichia coli. This approach was found to identify 41 ± 2 of the predicted 47 isoaccepting tRNAs in E. coli from targeted tandem mass spectrometry using only 24 precursor m/z values. This method should be easily adapted to other bacterial systems for both genomic and posttranscriptional analysis of tRNAs, and is likely suitable for future clinical applications.

Global identification of transfer RNAs by liquid chromatography-mass spectrometry (LC-MS).

Transfer ribonucleic acid (tRNA) is the non-coding RNA that links the processes of gene transcription with protein translation. While tRNAs have, individually, been studied for many years, few approaches exist for the global identification of tRNAs at the RNA and posttranscriptional RNA levels. Previously our lab introduced the concept of signature enzymatic digestion products (SDPs) for tRNA identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). SDPs enable the direct determination of tRNA identity based on mass spectrometry detection of unique m/z values from enzymatic digestion products. Here we have examined the applicability of liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) for global identification of bacterial tRNAs via their SDPs using Escherichia coli as the model system. Optimal ultra high performance and high performance liquid chromatography (UPLC vs. HPLC) conditions were identified to address the hundreds of enzymatic digestion products present in the sample. The use of LC-MS/MS improves the accuracy of SDP assignments through confirming sequence information. The combination of mass unique SDP detection along with MS/MS sequencing yielded the identification of all tRNA families from E. coli and nearly doubles the number of specific SDPs detected over that previously obtained using MALDI-MS. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.