Subcellular mRNA kinetic modeling reveals nuclear retention as rate-limiting.

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to lung infection than wild-type animals and that single-nucleotide polymorphisms in and were associated with pneumococcal pneumonia in humans. The effect of IL-33 on infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling.

Clinically used broad-spectrum antibiotics compromise inflammatory monocyte-dependent antibacterial defense in the lung.

Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers.

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue.

Purpose: Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue.

Transcriptome analysis reveals involvement of thiopurine S-methyltransferase in oxidation-reduction processes.

Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data.

mRNA stability and mA are major determinants of subcellular mRNA localization in neurons.

For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs.

CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown.

Loss of macpΨ Ribosomal RNA Modification Is a Major Feature of Cancer.

The ribosome is an RNA-protein complex that is essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive.

The human ZC3H3 and RBM26/27 proteins are critical for PAXT-mediated nuclear RNA decay.

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1.

Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis.

Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and associated factors. The effect of posttranslational modifications has not been studied systematically.

Systematic Detection of Poly(A) RNA-Interacting Proteins and Their Differential Binding.

RNA-binding proteins are dynamic posttranscriptional regulators of gene expression. Identification of mRNA-binding proteins in a given experimental setting is thus of great importance. We describe a procedure to enrich for direct poly(A) RNA protein binders by 4-thiouridine-enhanced UV cross-linking and oligo(dT) purification.

Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP.

PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP).

DDX54 regulates transcriptome dynamics during DNA damage response.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR).

Association of ITPA genotype with event-free survival and relapse rates in children with acute lymphoblastic leukemia undergoing maintenance therapy.

Although the treatment of acute lymphoblastic leukemia (ALL) has improved significantly over recent decades, failure due to treatment-related toxicities and relapse of the disease still occur in about 20% of patients. This retrospective study included 308 pediatric ALL patients undergoing maintenance therapy and investigated the effects of genetic variants of enzymes involved in the 6-mercaptopurine (6-MP) metabolism and folate pathway on survival and relapse rates. The presence of at least one of the non-functional ITPA alleles (94C>A and/or IVS2+21A>C variant) was associated with longer event-free survival compared to patients with the wild-type ITPA genotype (p = 0.

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins.

Post-translational stabilization of thiopurine S-methyltransferase by S-adenosyl-L-methionine reveals regulation of TPMT*1 and *3C allozymes.

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.

Transcriptome-wide analysis of protein-RNA interactions using high-throughput sequencing.

Protein-RNA interactions are emerging as an important functional element in the regulation of gene expression. Cross-linking of proteins to RNA by UV irradiation followed by immunoprecipitation (CLIP) has provided a crucial tool for research in this field. Initially, the bottleneck of the method was the relatively low number of identified RNA binding sites.

MTHFR and TYMS genotypes influence TPMT activity and its differential modulation in males and females.

Objectives: TPMT catalyzes the deactivation of the cytostatic drug 6-mercaptopurine used in treatment of cancer. Patients with low levels of TPMT more often experience severe toxic effects when treated with standard doses of 6-mercaptopurine than patients with high levels who, in turn, may experience inadequate treatment. It is therefore very important to assess factors that could influence TPMT activity.

S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts.

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity. We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT).

5,10-Methylenetetrahydrofolate reductase (MTHFR) low activity genotypes reduce the risk of relapse-related acute lymphoblastic leukemia (ALL).

The reported correlation of defects in 5,10-methylenetetrahydrofolate reductase (MTHFR), the key enzyme of folate metabolism, with modulated risk for acute lymphoblastic leukemia (ALL) is ambiguous. We have elucidated the influence of MTHFR genotype on ALL development and relapse in 140 Slovenian pediatric ALL patients and 183 healthy controls. A decreased proportion of low activity MTHFR genotypes was found in a group of ALL patients with relapses compared to healthy controls (p = 0.

Surface active stabilizer tyloxapol in colloidal dispersions exerts cytostatic effects and apoptotic dismissal of cells.

Solid lipid nanoparticles (SLN) have been praised for their advantageous drug delivery properties such as biocompatibility, controlled release and passive drug targeting. However, the cytotoxicity of SLN and their ingredients, especially over a longer time period, has not been investigated in detail. We examined the critical issues regarding the use of a surface active stabilizer Tyloxapol (Tyl) for the preparation of solid lipid particles (SLP) and their effects on cellular functions and viability.

A novel gene delivery system for stable transfection of thiopurine-S-methyltransferase gene in versatile cell types.

A novel gene delivery system termed artificial viral particles (AVPs) containing a plasmid coding for a recombinant fusion protein of enhanced green fluorescent protein (EGFP) with thiopurine-S-methyltransferase (TPMT) was designed for transfection of selected cell lines to establish stable clones which express recombinant EGFP-TPMT protein for further in vitro investigation of toxic effect of thiopurine drugs. Various AVPs based on a complex of the cationic polymer polyethylenimine (PEI) and anionic liposomes were formulated and transfection conditions were adapted in order to transfect the human Jurkat, HepG2 and HEK 293 cell lines. An adequate transfection rate was achieved with AVP containing branched low molecular weight PEI at a PEI:DNA charge ratio of 4.