Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae.

The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.

The Great Escape: mRNA Export through the Nuclear Pore Complex.

Nuclear export of messenger RNA (mRNA) through the nuclear pore complex (NPC) is an indispensable step to ensure protein translation in the cytoplasm of eukaryotic cells. mRNA is not translocated on its own, but it forms ribonuclear particles (mRNPs) in association with proteins that are crucial for its metabolism, some of which; like Mex67/MTR2-NXF1/NXT1; are key players for its translocation to the cytoplasm. In this review, I will summarize our current body of knowledge on the basic characteristics of mRNA export through the NPC. To be granted passage, the mRNP cargo needs to bind transport receptors, which facilitate the nuclear export. During NPC transport, mRNPs undergo compositional and conformational changes. The interactions between mRNP and the central channel of NPC are described; together with the multiple quality control steps that mRNPs undergo at the different rings of the NPC to ensure only proper export of mature transcripts to the cytoplasm. I conclude by mentioning new opportunities that arise from bottom up approaches for a mechanistic understanding of nuclear export.

The functional complexity of the RNA-binding protein Yra1: mRNA biogenesis, genome stability and DSB repair.

The mRNA export adaptor Yra1 is essential in S. cerevisiae, and conserved from yeast to human (ALY/REF). It is well characterized for its function during transcription elongation, 3' processing and mRNA export. Recently, different studies linked Yra1 to genome stability showing that Yra1 overexpression causes DNA Double Strand Breaks through DNA:RNA hybrids stabilization, and that Yra1 depletion affects DSB repair. However, the mechanisms through which Yra1 contributes to genome stability maintenance are not fully understood. Interestingly, our results showed that the Yra1 C-box domain is required for Yra1 recruitment to an HO-induced irreparable DSB following extensive resection, and that it is essential to repair an HO-induced reparable DSB. Furthermore, we defined that the C-box domain of Yra1 plays a crucial role in DSB repair through homologous recombination but not through non-homologous end joining. Future studies aim at deciphering the mechanism by which Yra1 contributes to DSB repair by searching for Yra1 partners important for this process. This review focuses on the functional complexity of the Yra1 protein, not only summarizing its role in mRNA biogenesis but also emphasizing its auto-regulation and implication in genome integrity either through DNA:RNA hybrids stabilization or DNA double strand break repair in S. cerevisiae.

Nucleocytosolic mRNA transport in plants: export factors and their influence on growth and development.

In eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.

Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.

A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

ALY proteins participate in multifaceted Nep1Mo-triggered responses in Nicotiana benthamiana and Arabidopsis thaliana.

Previously, it was found that Nep1Mo (a Nep1-like protein from Magnaporthe oryzae) could trigger a variety of plant responses, including stomatal closure, hypersensitive cell death (HCD), and defence-related gene expression, in Nicotiana benthamiana. In this study, it was found that Nep1Mo-induced cell death could be inhibited by the virus-induced gene silencing of NbALY916 in N. benthamiana. NbALY916-silenced plants showed impaired Nep1Mo-induced stomatal closure, decreased Nep1Mo-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced Nep1Mo-induced resistance against Phytophthora nicotianae. It also found that the deletion of AtALY4, an orthologue of NbALY916 in Arabidopsis thaliana, impaired Nep1Mo-triggered stomatal closure, HCD, and defence-related gene expression. The compromised stomatal closure observed in the NbALY916-silenced plants and AtALY4 mutants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor), and both Nep1Mo and H2O2 stimulated guard cell NO synthesis. Conversely, NO-induced stomatal closure was found not to require H2O2 synthesis; and NO treatment did not induce H2O2 production in guard cells. Taken together, these results demonstrate that the NbAlY916/AtAlY4-H2O2-NO pathway mediates multiple Nep1Mo-triggered responses, including stomatal closure, HCD, and defence-related gene expression.

A novel blood-based bioassay to monitor adiponectin signaling.

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

ALYREF-mediated RNA 5-Methylcytosine modification Promotes Hepatocellular Carcinoma Progression Via Stabilizing EGFR mRNA and pSTAT3 activation.

5-Methylcytosine (m5C) is one of the most ubiquitous modifications of mRNA and contributes to cancer pathogenesis. Aly/REF export factor (ALYREF), an m5C reader, is associated with the prognosis of liver hepatocellular carcinoma (LIHC). However, the effects of ALYREF on the progression of LIHC and the underlying molecular mechanisms remains elusive. Through an analysis of an online database and 3 independent LIHC cohorts, we found that ALYREF was markedly elevated in human liver cancer tissues and was significantly correlated with LIHC clinicopathological parameters, including Ki67+ cell rate, high-grade TNM stage, and poor prognosis. Several experiments were conducted to investigate the molecular basis and functional role of ALYREF-related progression in this study. ALYREF could enhance LIHC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and tumor formation in vivo. Mechanistically, ALYREF promoted the progression of human LIHC through EGFR pathways. Furthermore, ALYREF could directly bind to the m5C modification site of EGFR 3' untranslated region (3' UTR) to stabilize EGFR mRNA. Collectively, ALYREF played a crucial oncogenic role in LIHC via the stabilization of EGFR mRNA and subsequent activation of the STAT3 signaling pathway. Our results may help to elucidate the potential mechanisms of ALYREF-induced m5C modification in the progression of human LIHC.

The Adiponectin Receptor Agonist, ALY688: A Promising Therapeutic for Fibrosis in the Dystrophic Muscle.

Duchenne muscular dystrophy (DMD) is one of the most devastating myopathies, where severe inflammation exacerbates disease progression. Previously, we demonstrated that adiponectin (ApN), a hormone with powerful pleiotropic effects, can efficiently improve the dystrophic phenotype. However, its practical therapeutic application is limited. In this study, we investigated ALY688, a small peptide ApN receptor agonist, as a potential novel treatment for DMD. Four-week-old mdx mice were subcutaneously treated for two months with ALY688 and then compared to untreated mdx and wild-type mice. In vivo and ex vivo tests were performed to assess muscle function and pathophysiology. Additionally, in vitro tests were conducted on human DMD myotubes. Our results showed that ALY688 significantly improved the physical performance of mice and exerted potent anti-inflammatory, anti-oxidative and anti-fibrotic actions on the dystrophic muscle. Additionally, ALY688 hampered myonecrosis, partly mediated by necroptosis, and enhanced the myogenic program. Some of these effects were also recapitulated in human DMD myotubes. ALY688's protective and beneficial properties were mainly mediated by the AMPK-PGC-1α axis, which led to suppression of NF-κß and TGF-ß. Our results demonstrate that an ApN mimic may be a promising and effective therapeutic prospect for a better management of DMD.

Size matters in RNA export.

In eukaryotic cells, many RNA species are exported from the nucleus to the cytoplasms. Different RNA species form distinct ribonucleoprotein (RNP) complexes for export, indicating specific RNA recognition by export proteins. Specific RNA recognition is usually achieved by specific RNA sequences or structures, but we have recently reported a molecular mechanism by which the formation of export RNP complexes is specified by RNA length. ( 1) RNA polymerase II (Pol II) synthesizes not only mRNAs but also shorter RNAs, including spliceosomal U snRNAs. Although the key U snRNA export factor, PHAX, can bind to mRNA in vitro, PHAX is excluded from mRNA in vivo. The heterotetramer of the heterogeneous nuclear RNP (hnRNP) C1/C2 specifically binds Pol II transcripts longer than 200-300 nt, and funnels them into the mRNA export pathway by inhibiting their binding by PHAX, whereas shorter transcripts not bound by the heterotetramer are committed to the U snRNA export pathway. Although this finding reveals a novel function of the C1/C2 heterotetramer and highlights the biological importance of RNA recognition by length, it has raised a number of new questions, some of which will be discussed in this article, together with some historical background of this finding.

Novel insights into the role of 5-Methylcytosine RNA methylation in human abdominal aortic aneurysm.

Background: It remains largely unclear about the function of 5-methylcytosine (m5C) RNA modification in the context of abdominal aortic aneurysm (AAA). In this regard, the present work focused on investigating m5C RNA methylation and related modulator expression levels in AAA. Materials and methods: To this end, we quantified the m5C methylation levels in AAA tissues (n = 32) and normal aortic tissues (n = 12) to examine the mRNA m5C status and m5C modulator expression at mRNA and protein levels. Meanwhile, modulator localization within AAA tissue samples was detected by immunohistochemistry (IHC). Moreover, RNA immunoprecipitation-sequencing (RIP-seq) was also used to analyze the lncRNAs and mRNA binding to Aly/REF, as an m5C reader. Results: m5C expression markedly elevated in AAA in comparison with normal aortic samples in the AAA cases. The major 5-methylcytosine modulators, including NSUN2, NSUN5, and Aly/REF, which represented the major parameters related to the abnormal m5C modification level, were observed up-regulating in AAA tissues at both protein and mRNA levels. In addition, NSUN2 mRNA level remarkably related to Aly/REF expression, and they were co-expressed in the same cells in AAA group. Regarding the cellular location, Aly/REF was associated with inflammatory (CD45+, CD3+) infiltrates. Simultaneously, after screening for reads in AAA tissue compare with anti-Aly/REF group relative to IgG as control, we obtained totally 477 differentially expressed Aly/REF-binding lncRNAs and 369 differentially expressed Aly/REF-binding mRNAs in AAA tissue. The functions of Aly/REF-interacting lncRNA were involved in immune system process and macrophages infiltration. Through regulatory network (lncRNA-mRNA) analysis, our findings predicted the potential mechanism of Aly/REF-induced lncBCL2L1 and Aly/REF-lncFHL1 axis in AAA and inspire the understanding of m5C and lncRNA in AAA. Conclusions: This study is the first to examine m5A modification within human AAA samples. Our results indicate that m5C modulators, namely, Aly/REF and NUSN2, play vital parts in the human AAA pathogenic mechanism, which shed new lights on the function of m5C modification within AAA. Taken together, findings in this work offer a possible RNA methylation modification mechanism within clinical AAA.

What causes an RNA-DNA hybrid to compromise genome integrity?

Transcription is a source of genome instability that stimulates mutation and recombination. Part of the damage produced by transcription is mediated by R-loops, non-B DNA structures that normally form by the re-annealing of the nascent RNA with the template DNA outside the catalytic center of the RNA polymerase, displacing the non-template strand. Recent discoveries have revealed that R-loops might not be harmful by themselves. Instead, chromatin compaction triggered by these structures seems necessary, as deduced from the histone modifications frequently found associated with harmful R-loops. Remarkably, hybrids may also become harmful if stabilized by specific RNA binding proteins, one example of which is the yeast Yra1. We discuss here the possible mechanisms by which cells may stabilize R-loops and the consequences on transcription-replication conflicts and telomere homeostasis.

Profiling microRNAs and their targets in an important fleshy fruit: tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) is an important and the most useful plant based diet. It is widely used for its antioxidant property. Presently, only two digits, tomato microRNAs (miRNAs) are reported in miRBase: a miRNA database. This study is aimed to profile and characterize more miRNAs and their targets in tomato. A comprehensive comparative genomic approach is applied and a total of 109 new miRNAs belonging to 106 families are identified and characterized from the tomato expressed sequence tags (ESTs). All these potential miRNAs are profiled for the first time in tomato. The profiled miRNAs are also observed with stable stem-loop structures (Precursor-miRNAs), whose length ranges from 45 to 329 nucleotides (nt) with an average of 125 nt. The mature miRNAs are found in the stem of pre-miRNAs and their length ranges from 19 to 24 nt with an average of 21 nt. Furthermore, twelve miRNAs are randomly selected and experimentally validated through RT-PCR. A total of 406 putative targets are also predicted for the newly 109 tomato miRNAs. These targets are involved in structural protein, metabolism, transcription factor, growth & development, stress related, signaling pathways, storage proteins and other vital processes. Some important proteins like; 9-cisepoxycarotenoid dioxygenase (NCED), transcription factor MYB, ATP-binding cassette transporters, terpen synthase, 14-3-3 and TIR-NBS proteins are also predicted as putative targets for tomato miRNAs. These findings improve a baseline data of miRNAs and their targets in tomato. This baseline data can be utilized to fine tune this important fleshy fruit for nutritional & antioxidant properties and also under biotic & abiotic stresses.

mRNA export: threading the needle.

After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress.