Layer Of Post-transcriptional Regulation Research Articles

MicroRNA-15a-5p acts as a tumor suppressor in histiocytosis by mediating CXCL10-ERK-LIN28a-let-7 axis

Erdheim–Chester disease (ECD) is characterized by excessive production and accumulation of histiocytes within multiple tissues and organs. ECD patients harbor recurrent mutations of genes associated with the RAS/RAF/MEK/ERK signaling pathway, particularly, the BRAFV600E mutation. Following our previous finding that miR-15a-5p is the most prominently downregulated microRNA in ECD patients compared to healthy individuals, we elucidated its role in ECD pathogenesis. Bioinformatics analysis followed by a luciferase assay showed that chemokine ligand 10 (CXCL10) is a target gene regulated by miRNA-15a-5p. This was confirmed in 24/34 ECD patients that had low expression of miR-15a-5p concurrent with upregulated CXCL10. Overexpression of miR-15a-5p in cell lines harboring BRAF or RAS mutations (Ba/F3, KG-1a and OCI-AML3) resulted in CXCL10 downregulation, followed by LIN28a and p-ERK signaling downregulation and let-7 family upregulation. Overexpression of miR-15a-5p inhibited cell growth and induced apoptosis by decreasing Bcl-2 and Bcl-xl levels. Analysis of sequential samples from 7 ECD patients treated with MAPK inhibitors (vemurafenib/cobimetinib) for 4 months showed miR-15a-5p upregulation and CXCL10 downregulation. Our findings suggest that miR-15a-5p is a tumor suppressor in ECD through the CXCL10-ERK-LIN28a-let7 axis, highlighting another layer of post-transcriptional regulation in this disease. Upregulation of miR-15a-5p in ECD patients may have a potential therapeutic role.

SWATH-MS based quantitive proteomics reveal regulatory metabolism and networks of androdioecy breeding system in Osmanthus fragrans

BackgroundThe fragrant flower plant Osmanthus fragrans has an extremely rare androdioecious breeding system displaying the occurrence of males and hermaphrodites in a single population, which occupies a crucial intermediate stage in the evolutionary transition between hermaphroditism and dioecy. However, the molecular mechanism of androdioecy plant is very limited and still largely unknown.ResultsHere, we used SWATH-MS-based quantitative approach to study the proteome changes between male and hermaphroditic O. fragrans pistils. A total of 428 proteins of diverse functions were determined to show significant abundance changes including 210 up-regulated and 218 down-regulated proteins in male compared to hermaphroditic pistils. Functional categorization revealed that the differentially expressed proteins (DEPs) primarily distributed in the carbohydrate metabolism, secondary metabolism as well as signaling cascades. Further experimental analysis showed the substantial carbohydrates accumulation associated with promoted net photosynthetic rate and water use efficiency were observed in purplish red pedicel of hermaphroditic flower compared with green pedicel of male flower, implicating glucose metabolism serves as nutritional modulator for the differentiation of male and hermaphroditic flower. Meanwhile, the entire upregulation of secondary metabolism including flavonoids, isoprenoids and lignins seem to protect and maintain the male function in male flowers, well explaining important feature of androdioecy that aborted pistil of a male flower still has a male function. Furthermore, nine selected DEPs were validated via gene expression analysis, suggesting an extra layer of post-transcriptional regulation occurs during O. fragrans floral development.ConclusionTaken together, our findings represent the first SWATH-MS-based proteomic report in androdioecy plant O. fragrans, which reveal carbohydrate metabolism, secondary metabolism and post-transcriptional regulation contributing to the androdioecy breeding system and ultimately extend our understanding on genetic basis as well as the industrialization development of O. fragrans.

Transcriptome-Wide Analysis of Interplay between mRNA Stability, Translation and Small RNAs in Response to Neuronal Membrane Depolarization

Experience-dependent changes to neural circuitry are shaped by spatially-restricted activity-dependent mRNA translation. Although the complexity of mRNA translation in neuronal cells is widely appreciated, translational profiles associated with neuronal excitation remain largely uncharacterized, and the associated regulatory mechanisms are poorly understood. Here, we employed ribosome profiling, mRNA sequencing and small RNA sequencing to profile transcriptome-wide changes in mRNA translation after whole cell depolarization of differentiated neuroblast cultures, and investigate the contribution of sequence-specific regulatory mechanisms. Immediately after depolarization, a functional partition between transcriptional and translational responses was uncovered, in which many mRNAs were subjected to significant changes in abundance or ribosomal occupancy, but not both. After an extended (2 h) post-stimulus rest phase, however, these changes became synchronized, suggesting that there are different layers of post-transcriptional regulation which are temporally separated but become coordinated over time. Globally, changes in mRNA abundance and translation were found to be associated with a number of intrinsic mRNA features, including mRNA length, GC% and secondary structures; however, the effect of these factors differed between both post-depolarization time-points. Furthermore, small RNA sequencing revealed that miRNAs and tRNA-derived small RNA fragments were subjected to peak changes in expression immediately after stimulation, during which these molecules were predominantly associated with fluctuations in mRNA abundance, consistent with known regulatory mechanisms. These data suggest that excitation-associated neuronal translation is subjected to extensive temporal coordination, with substantial contributions from a number of sequence-dependent regulatory mechanisms.

MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium.

Intestinal stem cells (ISCs) fuel the lifelong self‐renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA‐binding protein is crucial for the maintenance of the Lgr5 + ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator‐activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5 + stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A‐mediated regulation. We also show that high PPARγ signalling impairs Lgr5 + ISC function, thus uncovering a new layer of post‐transcriptional regulation that critically contributes to intestinal homeostasis.

Emerging Roles of Heat-Induced circRNAs Related to Lactogenesis in Lactating Sows.

Heat stress negatively influences milk production and disrupts normal physiological activity of lactating sows, but the precious mechanisms by which hyperthermia adversely affects milk synthesis in sows still remain for further study. Circular RNAs are a novel class of non-coding RNAs with regulatory functions in various physiological and pathological processes. The expression profiles and functions of circRNAs of sows in lactogenesis remain largely unknown. In the present study, long-term heat stress (HS) resulted in a greater concentration of serum HSP70, LDH, and IgG, as well as decreased levels of COR, SOD, and PRL. HS reduced the total solids, fat, and lactose of sow milk, and HS significantly depressed CSNαs1, CSNαs2, and CSNκ biosynthesis. Transcriptome sequencing of lactating porcine mammary glands identified 42 upregulated and 25 downregulated transcripts in HS vs. control. Functional annotation of these differentially-expressed transcripts revealed four heat-induced genes involved in lactation. Moreover, 29 upregulated and 21 downregulated circRNA candidates were found in response to HS. Forty-two positively correlated circRNA-mRNA expression patterns were constructed between the four lactogenic genes and differentially expressed circRNAs. Five circRNA-miRNA-mRNA post-transcriptional networks were identified involving genes in the HS response of lactating sows. In this study we establish a valuable resource for circRNA biology in sow lactation. Analysis of a circRNA-miRNA-mRNA network further uncovered a novel layer of post-transcriptional regulation that could be used to improve sow milk production.

Human ALKBH3-induced m1A demethylation increases the CSF-1 mRNA stability in breast and ovarian cancer cells

In ovarian and breast cancers, the actions of the cytokine CSF-1 lead to poor prognosis. CSF-1 expression can be regulated post-transcriptionally. RNA methylation is another layer of posttranscriptional regulation. The methylation of N1 atom of adenine (m1A) results in a conformational change of RNA which regulates translational efficiency. Our study indicates that the m1A is also involved in the CSF-1 mRNA decay. The alteration of ALKBH3 expression, an m1A demethylase, regulates the CSF-1 mRNA stability. Demethylation of m1A by ALKBH3 increases the half-life of CSF-1 mRNA without affecting the translation efficiency. The m1A in CSF-1 mRNA is mapped in the 5′UTR near the translation initiation site. YTHDF2, a known m6A reader which interacts with the CCR4-NOT deadenylation complex, is not the reader of m1A-containing CSF-1 mRNA. Overexpression of ALKBH3 increases CSF-1 expression and the degree of cancer cell invasiveness without affecting cell proliferation or migration. Collectively, we showed that CSF-1 mRNA decay can be regulated at an epigenetic level, and that alteration of the N1‑methylation status leads to phenotypic changes in cancer cell behavior.

Regulatory landscape of the Hox transcriptome.

Precise regulation of Hox gene activity is essential to achieve proper control of animal embryonic development and to avoid generation of a variety of malignancies. This is a multilayered process, including complex polycistronic transcription, RNA processing, microRNA repression, long noncoding RNA regulation and sequence-specific translational control, acting together to achieve robust quantitative and qualitative Hox protein output. For many such mechanisms, the Hox cluster gene network has turned out to serve as a paradigmatic model for their study. In this review, we discuss current knowledge of how the different layers of post-transcriptional regulation and the production of a variety of noncoding RNA species control Hox output, and how this shapes formation of developmental systems that are reproducibly patterned by complex Hox networks.

Environment-dependent regulation of spliceosome activity by the LSM2-8 complex in Arabidopsis

Spliceosome activity is tightly regulated to ensure adequate splicing in response to internal and external cues. It has been suggested that core components of the spliceosome, such as the snRNPs, would participate in the control of its activity. The experimental indications supporting this proposition, however, remain scarce, and the operating mechanisms poorly understood. Here, we present genetic and molecular evidence demonstrating that the LSM2–8 complex, the protein moiety of the U6 snRNP, regulates the spliceosome activity in Arabidopsis, and that this regulation is controlled by the environmental conditions. Our results show that the complex ensures the efficiency and accuracy of constitutive and alternative splicing of selected pre-mRNAs, depending on the conditions. Moreover, miss-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that the LSM2–8 complex also guarantees adequate levels of the corresponding functional transcripts. Interestingly, the selective role of the complex has relevant physiological implications since it is required for adequate plant adaptation to abiotic stresses. These findings unveil an unanticipated function for the LSM2–8 complex that represents a new layer of posttranscriptional regulation in response to external stimuli in eukaryotes.

Chapter Three - The miRNA-Mediated Cross-Talk between Transcripts Provides a Novel Layer of Posttranscriptional Regulation

Endogenously expressed transcripts that are posttranscriptionally regulated by the same microRNAs (miRNAs) will, in principle, compete for the binding of their shared small noncoding RNA regulators and modulate each other's abundance. Recently, the levels of some coding as well as noncoding transcripts have indeed been found to be regulated in this way. Transcripts that engage in such regulatory interactions are referred to as competitive endogenous RNAs (ceRNAs). This novel layer of posttranscriptional regulation has been shown to contribute to diverse aspects of organismal and cellular biology, despite the number of functionally characterized ceRNAs being as yet relatively low. Importantly, increasing evidence suggests that the dysregulation of some ceRNA interactions is associated with disease etiology, most preeminently with cancer. Here we review how posttranscriptional regulation by miRNAs contributes to the cross-talk between transcripts and review examples of known ceRNAs by highlighting the features underlying their interactions and what might be their biological relevance.

A Viral Genome Landscape of RNA Polyadenylation from KSHV Latent to Lytic Infection

RNA polyadenylation (pA) is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV) infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ∼90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site) in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3′RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A) signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6), K9 (vIRF1), K10.5 (vIRF3), K11 (vIRF2), K12 (Kaposin A), T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression.