Eukaryotic Ribonucleic Acid Research Articles

Characterizing Benzo[a]pyrene Adducts in Transfer RNAs Using Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS).

The activated forms of the environmental pollutant benzo[a]pyrene (B[a]P), such as benzo[a]pyrene diol epoxide (BPDE), are known to cause damage to genomic DNA and proteins. However, the impact of BPDE on ribonucleic acid (RNA) remains unclear. To understand the full spectrum of potential BPDE-RNA adducts formed, we reacted ribonucleoside standards with BPDE and characterized the reaction products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To understand the potential types of adducts that could form with biological RNAs, eukaryotic transfer RNAs (tRNAs) were also reacted with BPDE. The isolation and analysis of the modified and adducted ribonucleosides using LC-MS/MS revealed several BPDE derivatives of post-transcriptional modifications. The approach outlined in this work enables the identification of RNA adducts from BPDE, which can pave the way for understanding the potential impacts of such adducts on the higher-order structure and function of modified RNAs.

Modification of STIM2 by m6A RNA methylation inhibits metastasis of cholangiocarcinoma.

BackgroundN6-methyladenosine (m6A) is the most frequent internal methylation of eukaryotic RNA (ribonucleic acid) transcripts and plays an important function in RNA processing. The current research aimed to investigate the role of m6A-STIM2 axis in cholangiocarcinoma (CCA) progression.MethodsThe expression of STIM2 (Stromal Interaction Molecule 2) in CCA was measured using quantitative polymerase chain reaction (PCR) and immunohistochemistry (IHC). STIM2 was examined in vivo for its effects on the malignant phenotypes of CCA cells. The m6A modification of STIM2 was assessed through MeRIP (methylated RNA Immunoprecipitation)-PCR.ResultsBased on the GEPIA (Gene Expression Profiling Interactive Analysis) 2 database findings, a low STIM2 mRNA (messenger RNA) level was related to a poor prognosis in individuals with CCA. Quantitative PCR and IHC assays indicated decreased protein satin in CCA tissues and were associated with extrahepatic metastasis. Vianude mice tail vein injection model indicated that increased STIM2 levels suppressed CCA cell metastasis in vivo, while KRT8 (keratin 8) was detected as the direct downstream target of STIM2-mediated CCA cell metastasis in vivo. Meanwhile, based on SRAMP database and MeRIP assays indicated that m6A alteration resulted in abnormal STIM2 expression in CCA via METTL14 and YTHDC2.ConclusionsOur findings revealed the epi-transcriptomic dysregulation in CCA and metastasis by proposing a complicated STIM2-KRT8 regulatory paradigm based on m6A alteration.

MetamORF: a repository of unique short open reading frames identified by both experimental and computational approaches for gene and metagene analyses.

The development of high-throughput technologies revealed the existence of non-canonical short open reading frames (sORFs) on most eukaryotic ribonucleic acids. They are ubiquitous genetic elements conserved across species and suspected to be involved in numerous cellular processes. MetamORF (https://metamorf.hb.univ-amu.fr/) aims to provide a repository of unique sORFs identified in the human and mouse genomes with both experimental and computational approaches. By gathering publicly available sORF data, normalizing them and summarizing redundant information, we were able to identify a total of 1 162 675 unique sORFs. Despite the usual characterization of ORFs as short, upstream or downstream, there is currently no clear consensus regarding the definition of these categories. Thus, the data have been reprocessed using a normalized nomenclature. MetamORF enables new analyses at locus, gene, transcript and ORF levels, which should offer the possibility to address new questions regarding sORF functions in the future. The repository is available through an user-friendly web interface, allowing easy browsing, visualization, filtering over multiple criteria and export possibilities. sORFs can be searched starting from a gene, a transcript and an ORF ID, looking in a genome area or browsing the whole repository for a species. The database content has also been made available through track hubs at UCSC Genome Browser. Finally, we demonstrated an enrichment of genes harboring upstream ORFs among genes expressed in response to reticular stress. Database URL https://metamorf.hb.univ-amu.fr/

Extensive 5\u2032-surveillance guards against non-canonical NAD-caps of nuclear mRNAs in yeast

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5′-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3′-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome‐like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single‐stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum.

†RNA-like Structures in the Genomes of RNA Viruses

All structural studies of ribonucleic acids (RNAs) from various origins—be they cellular messenger RNAs or viral RNAs—show that these molecules contain nucleotide sequences that are not translated into polypeptides. At the 5' end, besides the “cap” structure generally found in eukaryotic mRNAs and viral RNAs, the untranslated sequence is heteropolymeric and of variable length—from a few nucleotides to a few hundred nucleotides. The physiological role of these untranslated heteropolymeric regions is not well understood. This chapter summarizes the results obtained with different viral RNAs that accept an amino acid and are recognized by various tRNA-specific enzymes. For a number of these viral RNAs, the nucleotide sequence of the amino-acid-acceptor regions has been determined and the structures have been compared. Results indicated that different primary and secondary structures of two nucleic acid molecules, such as tRNAs and certain viral RNAs, can be recognized efficiently by the same enzyme systems that play a central role in the transfer of genetic information. The nucleotide sequence of virtually all tRNA molecules can be drawn in a hydrogen-bonded “cloverleaf” structure. The existence of this structure, further folded into a tertiary structure, has been proved by X-ray diffraction analyses. The chapter concludes with a discussion on the possible role of tRNA-like structures in viral RNA genomes.

Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein.

Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed.