Sevoflurane exerts protection against myocardial ischemia-reperfusion injury and pyroptosis through the circular RNA PAN3/microRNA-29b-3p/stromal cell-derived factor 4 axis.

OBJECTIVE: Sevoflurane is suggested to exert protective functions against myocardial ischemia-reperfusion injury (MIRI). However, the particular mechanism remains elusive. Therefore, this research explored the mechanism of sevoflurane in MIRI-induced damage and pyroptosis. METHODS: Subsequent to gain-or loss-of-function assays or/and sevoflurane treatment, the MIRI model was developed in rats. Cardiac function and body and heart weight of rats were evaluated, followed by measurement of apoptosis and creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), and pyroptosis-related protein levels. After loss-of-function assays or/and sevoflurane treatment in human cardiomyocytes (HCMs), the hypoxia/reoxygenation (H/R) model was constructed. In HCMs, cell viability, apoptosis, and pyroptosis-related proteins were detected. Circular RNA PAN3 (circPAN3), microRNA (miR)-29b-3p, and stromal cell-derived factor 4 (SDF4) expression was determined in rat myocardial tissues and HCMs. Mechanistically, interactions among circPAN3, miR-29b-3p, and SDF4 were analyzed. RESULTS: MIRI modeling increased miR-29b-3p expression and diminished circPAN3 and SDF4 expression in H/R-treated HCMs and MIRI rats, which was nullified by sevoflurane preconditioning. Mechanistically, circPAN3 negatively targeted miR-29b-3p to upregulate SDF4. Moreover, sevoflurane preconditioning reduced heart weight/body weight ratio, LDH, CK-MB, myocardial infarct size, left ventricular end-diastolic pressure, apoptosis, and pyroptosis, while elevating the increase and decrease of left ventricular pressure (±dp/dt max) and left ventricular systolic pressure in MIRI rats. In addition, sevoflurane preconditioning augmented viability while diminishing apoptosis and pyroptosis in H/R-treated HCMs. Moreover, circPAN3 silencing or miR-29b-3p overexpression abrogated alleviatory effects of sevoflurane on myocardial injury and pyroptosis in vitro. CONCLUSION: Sevoflurane treatment ameliorated myocardial injury and pyroptosis in MIRI via circPAN3/miR-29b-3p/SDF4 axis.

Exploration of the shared pathways and common biomarker PAN3 in ankylosing spondylitis and ulcerative colitis using integrated bioinformatics analysis.

Background: Ulcerative colitis (UC) is a chronic autoimmune-related disease that causes inflammation of the intestine. Ankylosing spondylitis (AS) is a common extraintestinal complication of UC involving the sacroiliac joint. However, the pathogenesis of AS secondary to UC has not been studied. This study aimed to investigate the shared pathways and potential common biomarkers of UC and AS. Methods: Microarray data downloaded from the Gene Expression Omnibus (GEO) database were used to screen differentially expressed genes (DEGs) in the UC and AS datasets. Weighted gene co-expression network analysis (WGCNA) was performed to identify co-expression modules related to UC and AS. Shared genes were then further analyzed for functional pathway enrichment. Next, the optimal common biomarker was selected using SVM-RFF and further validated using two independent GEO datasets. Finally, immune infiltration analysis was used to investigate the correlation of immune cell infiltration with common biomarkers in UC and AS. Results: A total of 4428 and 2438 DEGs in UC and AS, respectively, were screened. Four modules were identified as significant for UC and AS using WGCNA. A total of 25 genes overlapped with the strongest positive and negative modules of UC and AS. KEGG analysis showed these genes may be involved in the mitogen-activated protein kinase (MAPK) signaling pathway. GO analysis indicated that these genes were significantly enriched for RNA localization. PAN3 was selected as the optimal common biomarker for UC and AS. Immune infiltration analysis showed that the expression of PAN3 was correlated with changes in immune cells. Conclusion: This study first explored the common pathways and genetic diagnostic markers involved in UC and AS using bioinformatic analysis. Results suggest that the MAPK signaling pathway may be associated with both pathogeneses and that PAN3 may be a potential diagnostic marker for patients with UC complicated by AS.

Pan-3D genome analysis reveals structural and functional differentiation of soybean genomes.

BACKGROUND: High-order chromatin structure plays important roles in gene regulation. However, the diversity of the three-dimensional (3D) genome across plant accessions are seldom reported. RESULTS: Here, we perform the pan-3D genome analysis using Hi-C sequencing data from 27 soybean accessions and comprehensively investigate the relationships between 3D genomic variations and structural variations (SVs) as well as gene expression. We find that intersection regions between A/B compartments largely contribute to compartment divergence. Topologically associating domain (TAD) boundaries in A compartments exhibit significantly higher density compared to those in B compartments. Pan-3D genome analysis shows that core TAD boundaries have the highest transcription start site (TSS) density and lowest GC content and repeat percentage. Further investigation shows that non-long terminal repeat (non-LTR) retrotransposons play important roles in maintaining TAD boundaries, while Gypsy elements and satellite repeats are associated with private TAD boundaries. Moreover, presence and absence variation (PAV) is found to be the major contributor to 3D genome variations. Nevertheless, approximately 55% of 3D genome variations are not associated with obvious genetic variations, and half of them affect the flanking gene expression. In addition, we find that the 3D genome may also undergo selection during soybean domestication. CONCLUSION: Our study sheds light on the role of 3D genomes in plant genetic diversity and provides a valuable resource for studying gene regulation and genome evolution.

Molecular Insights into mRNA Polyadenylation and Deadenylation.

Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and shortening of a poly(A) tail are regulated by large multiprotein complexes. However, the molecular mechanisms of these protein machineries still remain unclear. Recent studies regarding the structural and biochemical characteristics of those protein complexes have shed light on the potential mechanisms of polyadenylation and deadenylation. This review summarizes the recent structural studies on pre-mRNA 3'-end processing complexes that initiate the polyadenylation and discusses the similarities and differences between yeast and human machineries. Specifically, we highlight recent biochemical efforts in the reconstitution of the active human canonical pre-mRNA 3'-end processing systems, as well as the roles of RBBP6/Mpe1 in activating the entire machinery. We also describe how poly(A) tails are removed by the PAN2-PAN3 and CCR4-NOT deadenylation complexes and discuss the emerging role of the cytoplasmic poly(A)-binding protein (PABPC) in promoting deadenylation. Together, these recent discoveries show that the dynamic features of these machineries play important roles in regulating polyadenylation and deadenylation.

Pan2-Pan3 complex, together with Ccr4-Not complex, has a role in the cell growth on non-fermentable carbon sources.

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3' poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3'-5' RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.

Poly(A) tail degradation in human cells: ATF4 mRNA as a model for biphasic deadenylation.

Eukaryotic mRNA deadenylation is generally considered as a two-step process in which the PAN2-PAN3 complex initiates the poly(A) tail degradation while, in the second step, the CCR4-NOT complex completes deadenylation, leading to decapping and degradation of the mRNA body. However, the mechanism of the biphasic poly(A) tail deadenylation remains enigmatic in several points such as the timing of the switch between the two steps, the role of translation termination and the mRNAs population involved. Here, we have studied the deadenylation of endogenous mRNAs in human cells depleted in either PAN3 or translation termination factor eRF3. Among the mRNAs tested, we found that only the endogenous ATF4 mRNA meets the biphasic model for deadenylation and that eRF3 prevents the shortening of its poly(A) tail. For the other mRNAs, the poor effect of PAN3 depletion on their poly(A) tail shortening questions the mode of their deadenylation. It is possible that these mRNAs experience a single step deadenylation process. Alternatively, we propose that a very short initial deadenylation by PAN2-PAN3 is followed by a rapid transition to the second phase involving CCR4-NOT complex. These differences in the timing of the transition from one deadenylation step to the other could explain the difficulties encountered in the generalization of the biphasic deadenylation model.

Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for NTRK fusion cancers.

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (NTRK1, NTRK2 and NTRK3) genes, respectively. TRKs can regulate cell proliferation, differentiation and even apoptosis through the RAS/MAPKs, PI3K/AKT and PLCγ pathways. Gene fusions involving NTRK act as oncogenic drivers of a broad diversity of adult and pediatric tumors, and TRKs have become promising antitumor targets. Therefore, achieving a comprehensive understanding of TRKs and relevant TRK inhibitors should be urgently pursued for the further development of novel TRK inhibitors for potential clinical applications. This review focuses on summarizing the biological functions of TRKs and NTRK fusion proteins, the development of small-molecule TRK inhibitors with different chemotypes and their activity and selectivity, and the potential therapeutic applications of these inhibitors for future cancer drug discovery efforts.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

PABP Cooperates with the CCR4-NOT Complex to Promote mRNA Deadenylation and Block Precocious Decay.

Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>∼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the ∼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.

PAN3-PSMA2 fusion resulting from a novel t(7;13)(p14;q12) chromosome translocation in a myelodysplastic syndrome that evolved into acute myeloid leukemia.

BACKGROUND: Acquired primary chromosomal changes in cancer are sometimes found as sole karyotypic abnormalities. They are specifically associated with particular types of neoplasia, essential in establishing the neoplasm, and they often lead to the generation of chimeric genes of pathogenetic, diagnostic, and prognostic importance. Thus, the report of new primary cancer-specific chromosomal aberrations is not only of scientific but also potentially of clinical interest, as is the detection of their gene-level consequences. CASE PRESENTATION: RNA-sequencing was performed on a bone marrow sample from a patient with myelodysplastic syndrome (MDS). The karyotype was 46,XX,t(7;13)(p14;q12)[2]/46,XX[23]. The MDS later evolved into acute myeloid leukemia (AML) at which point the bone marrow cells also contained additional, secondary aberrations. The 7;13-translocation resulted in fusion of the gene PAN3 from 13q12 with PSMA2 from 7p14 to generate an out-of-frame PAN3-PSMA2 fusion transcript whose presence was verified by RT-PCR together with Sanger sequencing. Interphase fluorescence in situ hybridization analysis confirmed the existence of the chimeric gene. CONCLUSIONS: The novel t(7;13)(p14;q12)/PAN3-PSMA2 in the neoplastic bone marrow cells could affect two key protein complex: (a) the PAN2/PAN3 complex (PAN3 rearrangement) which is responsible for deadenylation, the process of removing the poly(A) tail from RNA, and (b) the proteasome (PSMA2 rearrangement) which is responsible for degradation of intracellular proteins. The patient showed a favorable response to decitabine after treatment with 5-azacitidine and conventional intensive chemotherapy had failed. Whether this might represent a consistent feature of MDS/AML with this particular gene fusion, remains unknown.

Analysis of mRNA deadenylation by multi-protein complexes.

Poly(A) tails are found at the 3' end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes. Here, we describe an in vitro deadenylation assay developed to study the exonucleolytic activities of the multi-protein Ccr4-Not and Pan2-Pan3 complexes. We discuss how this assay can be used with short synthetic RNAs, as well as longer RNA substrates generated using in vitro transcription. Importantly, quantitation of the reactions allows detailed analyses of deadenylation in the presence and absence of accessory factors, leading to new insights into targeted mRNA decay.

Antagonistic actions of two human Pan3 isoforms on global mRNA turnover.

Deadenylation is a fundamental process that regulates eukaryotic gene expression. Mammalian deadenylation exhibits biphasic kinetics, with the Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes mediating the first and second phase, respectively; however, the significance of the biphasic nature of deadenylation in mRNA turnover remains unclear. In this study, we discovered that two distinct isoforms of human Pan3 display opposing properties necessary for coordinating the two phases of deadenylation. The shorter isoform (Pan3S) interacts more strongly with PABP than the longer isoform (Pan3L) does. Pan2 deadenylase activity is enhanced by Pan3S but suppressed by Pan3L. Knocking down individual Pan3 isoforms has opposing effects on the global poly(A) tail length profile, P-body formation, and different mRNA decay pathways. Transcriptome-wide analysis of Pan3 knockdown effects on mRNA turnover shows that depleting either Pan3 isoform causes profound and extensive changes in mRNA stability globally. These results reveal a new fundamental step governing mammalian mRNA metabolism. We propose that the first phase of deadenylation, coordinated through the interplay among the two Pan3 isoforms, Pan2, and PABP, represents a cytoplasmic mRNA maturation step important for proper mRNA turnover.

Effects of miR-143 on invasion and migration of B cell lymphoma cells through regulating expression of PAN3 protein / 中国免疫学杂志

Objective:To investigate the effect of miR-143 on the invasion and migration of B cell lymphoma cells .Methods:The expression of miR-143 in normal bone marrow and lymphoma was detected by qPCR .The expression levels of miR-143 in different cell lines were examined by qPCR .qPCR was used to detect the ability of miR-143 on PAN3.The relationship between miR-143 and PAN3 was detected by double luciferase assay .The effect of miR-143 expression on the migration ability of B cell lymphoma E 6-1 cells was examined by scratch test .The effect of miR-143 expression on the invasion ability of B cell lymphoma E 6-1 cells was exa mined by transwell test.Results:Compared with normal bone marrow ,miR-143 was down-regulated in B-cell lymphoma.Double luciferase assay showed that miR-143 could regulate the expression of PAN 3.Overexpression of miR-143 ,E6-1 cells significantly reduced the ability to attack and migrate.Conclusion:miR-143 can regulate the migration and invasion of B cell lymphoma cells by regulating the expression of PAN3.

Arsenite inhibits mRNA deadenylation through proteolytic degradation of Tob and Pan3.

The poly(A) tail of mRNAs plays pivotal roles in the posttranscriptional control of gene expression at both translation and mRNA stability. Recent findings demonstrate that the poly(A) tail is globally stabilized by some stresses. However, the mechanism underlying this phenomenon has not been elucidated. Here, we show that arsenite-induced oxidative stress inhibits deadenylation of mRNA primarily through downregulation of Tob and Pan3, both of which mediate the recruitment of deadenylases to mRNA. Arsenite selectively induces the proteolytic degradation of Tob and Pan3, and siRNA-mediated knockdown of Tob and Pan3 recapitulates stabilization of the mRNA poly(A) tail observed during arsenite stress. Although arsenite also inhibits translation by activating the eIF2α kinase HRI, arsenite-induced mRNA stabilization can be observed under HRI-depleted conditions. These results highlight the essential role of Tob and Pan3 in the stress-induced global stabilization of mRNA.

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation.

The conserved eukaryotic Pan2-Pan3 deadenylation complex shortens cytoplasmic mRNA 3' polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2-Pan3 complex.