Validation strategies for antibodies targeting modified ribonucleotides.

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.

Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.

Interactions, localization, and phosphorylation of the m6A generating METTL3-METTL14-WTAP complex.

N6-methyladenine (m6A) is found on many eukaryotic RNAs including mRNAs. m6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates m6A on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.

Determination of enrichment factors for modified RNA in MeRIP experiments.

In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

The ribosome biogenesis factor yUtp23/hUTP23 coordinates key interactions in the yeast and human pre-40S particle and hUTP23 contains an essential PIN domain.

Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA-protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3΄ half of the snR30 snoRNA. Protein-protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation.

The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans.

During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.

Pycnogenol® French maritime pine bark extract in randomized, double-blind, placebo-controlled human clinical studies.

Pycnogenol® French maritime pine bark extract is a well-known and thoroughly studied patented extract from the bark of Pinus pinaster Ait. ssp. Atlantica. In 39 randomized double-blind, placebo-controlled (RDP) human clinical trials including 2,009 subjects, Pycnogenol® French maritime pine bark extract supplementation for two weeks to six months has been shown to beneficially affect cardiovascular health, chronic venous insufficiency, cognition, joint health, skin health, eye health, women's health, respiratory health and allergies, oral health and sports performance. The mechanisms of action that can explain the respective effects on different conditions in the human body are discussed as well. As investigated in several in vitro, in vivo and in clinical studies, Pycnogenol® French maritime pine bark extract showed antioxidative effects, anti-inflammatory abilities, beneficial effects on endothelial function and reinforcing effects on the extracellular matrix. The present review aims to give a comprehensive overview of currently available "gold standard" RDP trials of Pycnogenol®'s benefits across various health domains compared to placebo. In addition, some of the processes on which the presented effects of Pycnogenol® French maritime pine bark extract are based will be elucidated and discussed. This broad overview of RDP studies on Pycnogenol® in different health domains can be used as a basis for further research on applications and mechanisms of this unique French maritime pine bark extract.

Review of Clinical Effects and Presumed Mechanism of Action of the French Oak Wood Extract Robuvit.

Since ancient times, oak wood polyphenols are consumed concomitantly with beverages that are stored and aged in oak wood barrels. Among these polyphenols are roburins, which belong to the class of ellagitannins and only occur in oak. To date, water-extracted standardized French Quercus robur wood extract, commercially known as Robuvit®, has been investigated in 1172 subjects in over 20 published clinical trials. The results of the clinical studies are consistent with reported effects of urolithins regarding increased mitophagy, pointing to enhanced energy capacity. The Robuvit metabolite urolithin A, B, and C levels and the number of urolithin producers were found to be increased after intake of the extract. Mitophagy is a process, which assigns energy inefficient mitochondria to disassembly, followed by reconstruction to new and more efficient replacements. This effect of Robuvit was observed in different study groups. Supplementation of Robuvit is ascribed to aid chronically fatigued or burnt-out individuals to regain higher energy and activity levels. Robuvit has been further shown to improve conditions such as renal insufficiency, liver insufficiency, mild heart failure, posttraumatic stress disorder and fatigue after surgery and facilitate recovery from mild health impairments such as flu or hangover. There are also indications that Robuvit helps improve erectile function and general loss of vigor in elderly men. Ex vivo gene expression experiments using metabolites collected from Robuvit consumers point to increased ribosomal biogenesis in endothelial, neuronal, and keratinocyte cells. Higher ribosome density accelerates the peptide production to meet protein demand, making Robuvit a potential enhancer of physical endurance and performance. A study with recreational athletes, supplemented with Robuvit daily, reported significantly increased performance in triathlon.

Projected supportive effects of PycnogenolⓇ in patients suffering from multi-dimensional health impairments after a SARS-CoV2 infection.

Corona virus disease 2019 (COVID-19) is triggered by the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV2) and has rapidly developed into a worldwide pandemic. Unlike other SARS viruses, SARS-CoV2 does not solely impact the respiratory system, but additionally leads to inflammation of endothelial cells, microvascular injuries and coagulopathies, thereby affecting multiple organs. Recent reports of patients who were infected with SARS-CoV2 suggest persistent health problems even months after the initial infection. The French maritime pine bark extract PycnogenolⓇ has demonstrated anti-inflammatory, vascular and endothelium-protective effects in over 90 human clinical studies. It is proposed that PycnogenolⓇ may be beneficial in supporting recovery and mitigating symptoms and long-term consequences resulting from a SARS-CoV2 infection in COVID-19 patients.