Purification of Enzymatically Active Xrn1 for Removal of Non-capped mRNAs from In Vitro Transcription Reactions and Evaluation of mRNA Decapping Status In Vivo.

The cap is a 7-methylguanosine attached to the first messenger RNA (mRNA) nucleotide with a 5'-5' triphosphate bridge. This conserved eukaryotic modification confers stability to the transcripts and is essential for translation initiation. The specific mechanisms that govern transcript cytoplasmic longevity and translatability were always of substantial interest.

Expanding the Available RNA Labeling Toolbox With CutA Nucleotidyltransferase for Efficient Transcript Labeling with Purine and Pyrimidine Nucleotide Analogs.

RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates.

SKI complex: A multifaceted cytoplasmic RNA exosome cofactor in mRNA metabolism with links to disease, developmental processes, and antiviral responses.

RNA stability and quality control are integral parts of gene expression regulation. A key factor shaping eukaryotic transcriptomes, mainly via 3'-5' exoribonucleolytic trimming or degradation of diverse transcripts in nuclear and cytoplasmic compartments, is the RNA exosome. Precise exosome targeting to various RNA molecules requires strict collaboration with specialized auxiliary factors, which facilitate interactions with its substrates.

Towards superior mRNA caps accessible by click chemistry: synthesis and translational properties of triazole-bearing oligonucleotide cap analogs.

Messenger RNA (mRNA)-based gene delivery is a powerful strategy for the development of vaccines and therapeutics. Consequently, approaches that enable efficient synthesis of mRNAs with high purity and biological activity are in demand. Chemically modified 7-methylguanosine (mG) 5' caps can augment the translational properties of mRNA; however, efficient synthesis of structurally complex caps, especially on a large scale, is challenging.

2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am).

Reproducible and efficient new method of RNA 3'-end labelling by CutA nucleotidyltransferase-mediated CC-tailing.

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases.

Tricks and threats of RNA viruses - towards understanding the fate of viral RNA.

Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous. Their relatively simple structure makes the identification of viral invasion a difficult task for cells. In the course of evolution, viral nucleic acids have become one of the strongest and most reliable early identifiers of infection.

Elimination of 01/A'-A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3.

Pre-rRNA processing generates mature 18S, 5.8S, and 28S/25S rRNAs through multistage removal of surrounding 5'-ETS/3'-ETS and intervening ITS1/ITS2 segments. Endonucleolytic activities release by-products, which need to be eliminated.

Structural Characteristics of Simple RNA Repeats Associated with Disease and their Deleterious Protein Interactions.

Short Tandem Repeats (STRs) are frequent entities in many transcripts, however, in some cases, pathological events occur when a critical repeat length is reached. This phenomenon is observed in various neurological disorders, such as myotonic dystrophy type 1 (DM1), fragile X-associated tremor/ataxia syndrome, C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), and polyglutamine diseases, such as Huntington's disease (HD) and spinocerebellar ataxias (SCA). The pathological effects of these repeats are triggered by mutant RNA transcripts and/or encoded mutant proteins, which depend on the localization of the expanded repeats in non-coding or coding regions.

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0.

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA.

Purification of eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays.

Exoribonucleases-among the other RNases-play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specificity and optimal conditions for enzymatic action. One of the most significant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5'-end to 3'-end or in the opposite way.

RNA channelling by the eukaryotic exosome.

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome.

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L.

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine-subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p.