End Formation Research Articles

The emerging roles of CFIm25 (NUDT21/CPSF5) in human biology and disease.

The mammalian cleavage factor I subunit CFIm25 (NUDT21) binds to the UGUA sequences of precursor RNAs. Traditionally, CFIm25 is known to facilitate 3' end formation of pre-mRNAs resulting in the formation of polyadenylated transcripts. Recent studies suggest that CFIm25 may be involved in the cyclization and hence generation of circular RNAs (circRNAs) that contain UGUA motifs. These circRNAs act as competing endogenous RNAs (ceRNAs) that disrupt the ceRNA-miRNA-mRNA axis. Other emerging roles of CFIm25 include regulating both alternative splicing and alternative polyadenylation (APA). APA generates different sized transcripts that may code for different proteins, or more commonly transcripts that code for the same protein but differ in the length and sequence content of their 3' UTRs (3' UTR-APA). CFIm25 mediated global changes in 3' UTR-APA affect human physiology including spermatogenesis and the determination of cell fate. Deregulation of CFIm25 and changes in 3' UTR-APA have been implicated in several human diseases including cancer. In many cancers, CFIm25 acts as a tumor suppressor. However, there are some cancers where CFIm25 has the opposite effect. Alterations in CFIm25-driven 3' UTR-APA may also play a role in neural dysfunction and fibrosis. CFIm25 mediated 3' UTR-APA changes can be used to generate specific signatures that can be used as potential biomarkers in development and disease. Due to the emerging role of CFIm25 as a regulator of the aforementioned RNA processing events, modulation of CFIm25 levels may be a novel viable therapeutic approach. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease.

Revealing the key role of structural cross-link between lignin and polysaccharides during fast pyrolysis of lignocellulose

Lignin-carbohydrate complex (LCC) is the native existing form of major components in lignocellulose. In this study, the structural cross-link between lignin and polysaccharides in lignocellulose was quantitatively estimated with carboxymethylation-separation (CM-Sep) method, and its influence on lignocellulose pyrolysis was systematically investigated. The cross-linked lignin was found to positively correlate with the production of small molecules and furan derivatives while negatively affecting the generation of anhydrous sugars. Content of small molecules was increased by 97% while that of anhydrous sugars was decreased by 47% in pyrolytic products with levoglucosan yield lowered by 54 wt% in the existence of cross-linked lignin. Furthermore, the impact of cross-linked lignin was revealed to be significantly distinguished from free lignin. Impeded glycosidic end formation and boosted glycosyl ring scission as well as lignin fragmentation were responsible for the distinction. Excellent correlations between structural cross-link and lignocellulose pyrolytome could facilitate product prediction and process design.

СТАНОВЛЕНИЕ ГРАНИТОИДНЫХ (ГАББРО-ГРАНИТНЫХ) ПЛУТОНОВ И СВЯЗАННОГО С НИМИ ОРУДЕНЕНИЯ

Deep geological and geophysical modeling by a large number of granitoid plutons for the first time showed a completely different nature than was commonly believed. As a result, ideas about the connection of mineralization with them have changed dramatically. Many petrological contradictions and puzzles were solved logically and scientifically grounded. The complete and comprehensive review of the problem from the beginning (source of the initial magma) to the end (formation of the granite ore-magmatic system) is given. Three stages of the volatiles and ore matter concentration in the history of the plutons formation were established: the first (planetary) - during the intrachamber differentiation of the initial basalt magma with an isolation of granitoid melting; the second (regional) - during the formation of a residual leukogranite chamber the third, main (local) - during the intrusion of ore-generating apophyses from the chamber.

Raw Potato Starch Alters the Microbiome, Colon and Cecal Gene Expression, and Resistance to Citrobacter rodentium Infection in Mice Fed a Western Diet

ObjectivesTo evaluate the effects of feeding a Total Western Diet (TWD) based on NHANES data containing 0, 2, 5 or 10% raw potato starch on the microbiome, gene expression, and resistance to Citrobacter rodentium infection in mice.Methods3–4 week old C57Bl/6 mice were fed the TWD for 6 weeks followed by 3 weeks of the TWD containing 0, 3, 5, or 10% raw potato starch (RPS). Mice were euthanized and tissues collected for analysis. In other experiments, after 3 weeks on the RPS containing diets, mice were infected with Citrobacter rodenitium (Cr), mice euthanized at day 12 post-infection and tissues collected for analysis. DNA isolated from cecal or fecal contents was used for sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. For gene expression analysis, RNA isolated from colon and cecal tissues was sequenced on an Illumina HiSeq 4000 using a 1 × 50 bp single end format.ResultsFeeding the RPS diets resulted in changes to the microbiome with α-diversity decreasing with increasing levels of dietary RPS (p < 0.05). PCA analysis showed four discreet groupings based on the RPS level that were significantly different by 0.5 Unifrac PERMANOVA analysis (p < 0.001). The relative abundance of various genera was altered by feeding increasing levels of RPS. The genus Lachnospiraceae NK4A136 group was markedly increased in a dose-dependent fashion. Differential expression of the genes was highest in the cecum but decreased substantially in the proximal colon and further so in the distal colon. The pattern of gene expression observed suggest that RPS primes the intestine for immune responses to bacteria, parasites and viruses. However, mice fed the 10% RPS diet and infected with Cr had increased colonization of the colon at day 12 post-infection that was not observed at lower levels of dietary RPS (p < 0.05). Mice had increased hyperplasia and colonic pathology in the 10% RPS group compared to mice receiving diet without added RPS. 16S sequencing of cecal contents showed RPS-dependent changes in both uninfected and infected mice.ConclusionsDietary RPS significantly altered the microbiome of mice in the context of a NHANES-based Western Diet. Mice fed diets containing 10% RPS had significant changes in cecal and colon gene expression, Cr colon colonization and infection-induced pathology.Funding SourcesUSDA ARS.

Hierarchical length and sequence preferences establish a single major piRNA 3′-end

SummaryPIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of mobile genetic elements. PiRNAs use extensive base-pairing to recognize their targets and variable 3′ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3′end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference to cut in front of Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3′end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.

Identification of a Transferrable Terminator Element That Inhibits Small RNA Production and Improves Transgene Expression Levels.

The role of terminators is more commonly associated with the polyadenylation and 3′ end formation of new transcripts. Recent evidence, however, suggests that this regulatory region can have a dramatic impact on gene expression. Nonetheless, little is known about the molecular mechanisms leading to the improvements associated with terminator usage in plants and the different elements in a plant terminator. Here, we identified an element in the Arabidopsis HSP18.2 terminator (tHSP) to be essential for the high level of expression seen for transgenes under the regulation of this terminator. Our molecular analyses suggest that this newly identified sequence acts to improve transcription termination, leading to fewer read-through events and decreased amounts of small RNAs originating from the transgene. Besides protecting against silencing, the tHSP-derived sequence positively impacts splicing efficiency, helping to promote gene expression. Moreover, we show that this sequence can be used to generate chimeric terminators with enhanced efficiency, resulting in stronger transgene expression and significantly expanding the availability of efficient terminators that can be part of good expression systems. Thus, our data make an important contribution toward a better understanding of plant terminators, with the identification of a new element that has a direct impact on gene expression, and at the same time, creates new possibilities to modulate gene expression via the manipulation of 3′ regulatory regions.

Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the repertoire of proteins that govern isoform composition and abundance remains incomplete. Here, we show that the RNA kinase CLP1 regulates mRNA isoform expression through suppression of proximal cleavage and polyadenylation. We found that human stem-cell-derived motor neurons without CLP1 or with the disease-associated CLP1 p.R140H variant had distinct patterns of RNA-polymerase-II-associated cleavage and polyadenylation complex proteins that correlated with polyadenylation site usage. These changes resulted in imbalanced mRNA isoform expression of long genes important for neuronal function that were recapitulated invivo. Strikingly, we observed the same pattern of reduced mRNA isoform diversity in 3' end sequencing data from brain tissues of patients with neurodegenerative disease. Together, our results identify a previously uncharacterized role for CLP1 in mRNA 3' end formation and reveal an mRNA misprocessing signature in neurodegeneration that may suggest a common mechanism of disease.

Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3′ UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3′ end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Genetic and Biochemical Characterizations of aLhr1 Helicase in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

Homologous recombination (HR) refers to the process of information exchange between homologous DNA duplexes and is composed of four main steps: end resection, strand invasion and formation of a Holliday junction (HJ), branch migration, and resolution of the HJ. Within each step of HR in Archaea, the helicase-promoting branch migration is not fully understood. Previous biochemical studies identified three candidates for archaeal helicase promoting branch migration in vitro: Hjm/Hel308, PINA, and archaeal long helicase related (aLhr) 2. However, there is no direct evidence of their involvement in HR in vivo. Here, we identified a novel helicase encoded by Saci_0814, isolated from the thermophilic crenarchaeon Sulfolobus acidocaldarius; the helicase dissociated a synthetic HJ. Notably, HR frequency in the Saci_0814-deleted strain was lower than that of the parent strain (5-fold decrease), indicating that Saci_0814 may be involved in HR in vivo. Saci_0814 is classified as an aLhr1 under superfamily 2 helicases; its homologs are conserved among Archaea. Purified protein produced in Escherichia coli showed branch migration activity in vitro. Based on both genetic and biochemical evidence, we suggest that aLhr1 is involved in HR and may function as a branch migration helicase in S. acidocaldarius.

Apice closure in Pulp Revascularization Therapy. Case report and literature review

Background: Endodontic Regenerative Procedures, especially pulp revascularization therapy, have become a real option for the treatment for immature permanent teeth with open apices. This sort of approach has saved many teeth that otherwise would have been extracted. The technique is simple and effective, and can be accomplished by any odontologist. Objective: This article aimed to make a literature review to give support for a case report of an Endodontic Regenerative Procedure of a twelve-year-old male patient with immature open apice of tooth 21. Methodology: Articles were researched concerning pulp revascularization to reach root end formation. On line searches were accomplished, whose database include BVS/BIREME, Web of Science, PUBMED Central, Science Direct, Higher Level Personnel Improvement Coordinator (CAPES), The Cochrane Library, and PROSPERO). Results: the articles researched showed that Endodontic Regenerative Procedures are efficient in stimulating root end formation. The case report described matched the results offered by the articles, showing an immature open apice tooth that had its root end completely formed after pulp revascularization therapy. Conclusion: Pulp revascularization therapy is an efficient and practical treatment for immature teeth with open apices.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3′ end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3′ ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3′ ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3′ end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3′ end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

M6A RNA methylation facilitates pre-mRNA 3'-end formation and is essential for viability of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3’-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3’-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3’-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.

Modeling a human CLP1 mutation in mouse identifies an accumulation of tyrosine pre-tRNA fragments causing pontocerebellar hypoplasia type 10

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3′-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5′ tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules.

Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3' end formation in response to azithromycin.

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3' ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3' end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3' ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3' end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3' end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

Dynamics of capillary coalescence and breakup: Quasi-two-dimensional nematic and isotropic droplets.

We observed the formation of small satellite droplets from the bridge at droplet coalescence. Investigations were made using a Hele-Shaw cell in the two-phase region at the nematic-isotropic phase transition. In previous works on coalescence it was considered that before the start of coalescence there exists a bridge between the outer fluid connecting regions on the two sides of the droplets (outer bridge). After the start of coalescence, a bridge connecting the two droplets appears (droplet bridge) and the outer bridge is broken. We have shown that there are coalescence processes where after the start of coalescence both the droplet bridge and the outer bridge can exist. This cardinally changes the coalescence process. During the first coalescence stage the size of the outer bridge decreases and the size of the droplet bridge increases. During the second stage the outer bridge becomes unstable which leads to pinch-off, formation of pointed end domains, secondary instability, splitting of pointed end domains, and formation of satellite droplets. We found the linear dependence of the minimum bridge radius on time near bridge breakup. This behavior confirms the capillary viscous regime of bridge breakup. Our work connects two areas of fluid dynamics: coalescence and breakup with formation of satellite droplets.

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

A CRISPR RNA-Binding Protein Screen Reveals Novel Regulators of RUNX1 Alternative Polyadenylation

Alternative polyadenylation (APA) is a crucial post-transcriptional process that regulates the 3' end formation of over 75% of all human genes. Despite its prevalence and importance during development and in disease, there are few studies that focus on APA in the hematology field. Interestingly, APA affects the coding sequence of RUNX1, a pivotal transcription factor for proper hematopoiesis. Usage of the most proximal RUNX1 polyadenylation sequence (PAS) in upstream alternative terminal exon 7a results in the C-terminally truncated RUNX1a isoform. RUNX1a uniquely promotes self-renewal and expansion of hematopoietic stem cells (HSCs). In the endogenous context, usage of this proximal PAS is tightly restricted, resulting in polyadenylation at one of three distal PAS in exon 8 and producing the RUNX1b/c isoforms. RUNX1b/c are much more abundant and promote HSC differentiation and lineage commitment. Importantly, the ratio of RUNX1a to RUNX1b/c is developmentally-regulated. Therefore, it is highly significant to uncover the APA mechanism that controls the RUNX1 isoform ratio and consequently the balance of HSC self-renewal and differentiation. We hypothesize that there are enhancers and suppressors of the proximal RUNX1 PAS, tightly controlling the relative amount of RUNX1a produced during hematopoiesis.To identify RUNX1 APA regulators, we first developed a strategy to monitor RUNX1 proximal PAS usage in vitro. We cloned alternative terminal exon 7a, which contains the proximal PAS, along with 500 bp of up and downstream flanking intron between the two exons of a split GFP reporter. When we introduce this fluorescent minigene reporter into blood cells, two transcripts can be produced: (1) the first GFP exon splices and polyadenylates RUNX1 exon 7a, producing no GFP (RUNX1a formation) or (2) the two GFP exons are spliced together, skipping the proximal PAS in exon 7a, producing GFP (RUNX1b/c formation). Since RUNX1b/c are much more highly expressed than RUNX1a throughout hematopoiesis, we observed a robust GFP signal when this reporter was transfected into MDS-L, K562, and U937 cell lines. RT-PCR analysis confirmed that both transcripts are produced, albeit at dramatically different levels. We can thus use GFP fluorescence as a read-out for RUNX1 proximal PAS usage.Next, we generated a stable, clonal MDS-L cell line expressing this RUNX1 exon 7a split GFP reporter. We used this reporter line to perform a CRISPR screen using an sgRNA library that is enriched for targeting RNA-binding proteins (RBPs). We infected cells with the library, cultured in puromycin to force sgRNA and Cas9 integration, waited three weeks to ensure gene knockout, and then used fluorescence-activated cell sorting (FACS) to isolate GFP low and high cell populations. GFP low cells contained sgRNAs targeting RUNX1a suppressors since these cells exhibited enhanced proximal PAS usage. Conversely, GFP high cells contained sgRNAs targeting RUNX1a enhancers since proximal PAS usage was further diminished. We identified 44 putative RUNX1a suppressors and 41 putative enhancers (p < 0.05).We then sought to validate the genes revealed in our CRISPR screen by using individual sgRNAs targeting a subset of the candidates. One confirmed suppressor is HNRNPA1, an RBP which is upregulated and leukemogenic in BCR/ABL chronic myelogenous leukemia (CML). In HNRNPA1 knockout cells, there was a 5-fold increase in relative RUNX1a and a corresponding decrease in RUNX1b/c levels by RT-qPCR. This effect was dose-dependent as heterozygous HNRNPA1 cells exhibited an intermediate 2.5-fold increase in RUNX1a mRNA. We verified this data by performing shRNA knockdown of HNRNPA1 in MDS-L and K562 cells, again observing a significant increase in relative RUNX1a levels. Interestingly, HNRNPA1 is highly expressed throughout the course of hematopoiesis, indicating that a cooperating protein helps facilitate this suppressive effect. Indeed, numerous HNRNPA1-interacting proteins were also identified in our screen and are the subject of current studies.In summary, we devised a fluorescent minigene reporter to monitor RUNX1 APA in vitro. Using this reporter, we performed a CRISPR RBP screen and identified HNRNPA1 as the founding member of a complex that suppresses RUNX1a production during hematopoiesis. HNRNPA1 and additional regulators represent novel proteins that have the potential to regulate HSC behavior by modifying the RUNX1 isoform ratio. DisclosuresNo relevant conflicts of interest to declare.

Advanced glycation end products and their receptors in serum of patients with type 2 diabetes

Glycation is a non-enzymatic process involving the reaction of reducing sugars or reactive oxoaldehyde with proteins, lipids or nucleic acids, which results in the formation of advanced glycation end products (AGEs). The presented work discusses the glycation process in people with advanced stage of type 1 or type 2 diabetes. The concentration of different AGEs and their receptors for 58 serum samples was determined by ELISA and by spectrofluorimetric methods. In addition to fluorescent low molecular weight and protein-bound AGEs, we have also marked a new class of AGEs: melibiose-derived glycation product (MAGE). Our attention was also focused on the two groups of AGEs receptors: scavenger receptors (SR-A and SR-B) and RAGE. The correlation between the SR-AI scavenging receptors concentration and the fluorescence of AGEs as well as diabetes biological markers: GFR, creatinine contentration and HbA1c was demonstrated. A relationship between the concentration of AGEs and their receptors was also found in serum sample of patients treated with the metformin and aspirin. Furthermore, the concentration of SR-AI scavenger and the fluorescence of total AGEs was significantly lower in treated patients than in non treated patients. AGEs have also been found to contribute to the development of cardiovascular disease, atherosclerosis and diabetic complications, what could be deduced from the correlation of AGEs level and HDL cholesterol or uric acid level. Thus, it was confirmed that AGEs are involved in the pathomechanism of diabetes and other degenerative diseases. Nowadays, it is believed that AGEs due to the long time remaining in the body may be an important diagnostic marker. Their determination may allow monitoring the progression of the disease and the effectiveness of the therapy.

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.

CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma.

CircRNAs play essential roles in various physiological processes and involves in many diseases, in particular cancer. Global downregulation of circRNA expression has been observed in hepatocellular carcinoma (HCC) in many studies. Previous studies revealed that the pre-mRNA 3' end processing complex participates in circRNA cyclization and plays an important role in HCC tumorigenesis. Therefore, we explored the role of CPSF4, for 3' end formation and cleavage, in circRNA formation. Clinical research has shown that CPSF4 expression is upregulated in HCC and that high expression of CPSF4 is associated with poor prognosis in HCC patients. Mechanistic studies have demonstrated that CPSF4 reduces the levels of circRNAs, which possess a polyadenylation signal sequence and this decrease in circRNAs reduces the accumulation of miRNA and disrupts the miRNA-mediated gene silencing in HCC. Experiments in cell culture and xenograft mouse models showed that CPSF4 promotes the proliferation of HCC cells and enhances tumorigenicity. Moreover, CPSF4 antagonizes the tumor suppressor effect of its downstream circRNA in HCC. In summary, CPSF4 acts as an oncogene in HCC through circRNA inhibition and disruption of miRNA-mediated gene silencing.