Regulation Of mRNA Processing Research Articles

RNA modifications in insects.

More than 100 RNA chemical modifications to cellular RNA have been identified. N 6-methyladenosine (m6A) is the most prevalent modification of mRNA. RNA modifications have recently attracted significant attention due to their critical role in regulating mRNA processing and metabolism. tRNA and rRNA rank among the most heavily modified RNAs, and their modifications are essential for maintaining their structure and function. With our advanced understanding of RNA modifications, increasing evidence suggests RNA modifications are important in regulating various aspects of insect life. In this review, we will summarize recent studies investigating the impact of RNA modifications in insects, particularly highlighting the role of m6A in insect development, reproduction, and adaptation to the environment.

M6A demethylase OsALKBH5 is required for double-strand break formation and repair by affecting mRNA stability in rice meiosis.

N6-methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo-cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P-bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double-strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5-mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post-transcriptional regulation of meiotic progression in rice.

Abstract 3908: M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer

Abstract BACKGROUND: Chemotherapy resistance is recognized to occur not only through selection of pre-existing genetically resistant clones, but also through rapid phenotypic plasticity mechanisms. We previously reported that bladder cancer cells can rapidly transition to and from a chemo-resistant phenotype. Recently, N6-methyladenosine (m6A) RNA modifications were shown to dynamically and reversibly regulate mRNA processing, differentiation, and cell fate. Here, we tested the hypothesis that m6A modifications regulate phenotypic plasticity and transition to cisplatin resistance in bladder cancer (BC). METHODS: We utilized methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) and by RNA-seq to identify transcripts that were both differentially methylated and differentially expressed between cisplatin-sensitive and cisplatin-resistant BC cell lines. Candidate transcripts were annotated using Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Clinical relevance was evaluated using databases such as The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Cancer-relevant genes were then validated in vitro by targeted immunoprecipitation and PCR (MeRIP-PCR) and qPCR. Candidate genes were then functionally validated in cell lines and in patient derived organoids (PDOs) by siRNA-mediated knockdown and cisplatin treatment, as well as by m6A measurement after short-term cisplatin treatment. RESULTS: MeRIP-seq and RNA-seq revealed that cisplatin-sensitive and cisplatin-resistant BC cells have distinct m6A profiles, with 130 transcripts that were both differentially methylated and differentially expressed. Using clinical and functional database tools, we filtered this list to 37 transcripts and ranked them based on clinical relevance in BC. Of the top 15 candidates, eight were successfully validated in vitro via repeat qPCR and MeRIP-PCR. In cisplatin-resistant cells, one candidate, SLC7A11, was found to have decreased m6A level, which was associated with increased RNA stability and elevated transcript and protein levels. Consistent with this, 48 hr cisplatin treatment of cisplatin-sensitive cells led to SLC7A11 hypomethylation, increased RNA stability, and elevated transcript and protein levels. Furthermore, depletion of SLC7A11 by siRNA-knockdown re-sensitized BC cell lines and PDOs to cisplatin. CONCLUSIONS: Using unbiased transcriptome-wide m6A profiling followed by targeted validation, we demonstrated that m6A modifications regulate expression of clinically relevant gene transcripts in BC, and that a subset of these modifications may promote resistance to chemotherapy Collectively, these results implicate epitranscriptomic plasticity as a driver in potentiating cisplatin resistance in bladder cancer. Citation Format: Emmanuelle Hodara, Aubree Mades, Tong Xu, Amir Goldkorn, Suhn Rhie. M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3908.

CircPTPN12 promotes the progression and sunitinib resistance of renal cancer via hnRNPM/IL-6/STAT3 pathway

Renal cell carcinoma (RCC) is characterized by the difficulties in early diagnosis and the propensity to metastases. For advanced RCC, sunitinib targeted therapy is the clinically recommended first-line drug and the major challenge of sunitinib treatment is adaptive resistance. Therefore, it is imperative to research the mechanisms underlying sunitinib resistance. In this study, we discovered that circPTPN12 was highly expressed in RCC tissues and was associated with poorer clinical outcomes. circPTPN12 could promote the proliferation, migration, invasion, and sunitinib resistance of RCC cells. Mechanistically, circPTPN12 was found to form a complex with hnRNPM, which was involved in the regulation of mRNA processing. The combination with circPTPN12 enhanced the ability of hnRNPM to maintain the stability of IL-6 mRNA and further activated the STAT3 signaling pathway. The study revealed that circPTPN12/hnRNPM/IL-6/STAT3 axis promoted RCC progression and sunitinib resistance, which might be a promising therapeutic target for relieving sunitinib resistance in RCC.

Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato

The RanBP2-type zinc finger (RBZ) protein genes, which are well-characterized in animals, are involved in the regulation of mRNA processing. Although they are diversely distributed in plants, their functions still remain largely unknown. In this study, we performed a comprehensive bioinformatic analysis of 22 RBZ genes in tomato. The gene structure analysis revealed that the SlRBZ genes have 2 to 17 exons. SlRBZ proteins contain typical conserved domains, including Motif 1 or Motif 2, or a combination of Motif 9 and Motif 4. Two paralogous pairs were identified in the tomato. Segmental duplication possibly contributed to the expansion of the SlRBZ genes in tomato. Interestingly, the SlRBZ15 gene generated four products, yielded by alternative splicing. A cis-regulatory element analysis revealed that SlRBZ genes might be involved in the complex regulatory networks during plant growth and development. The expression profiles of the SlRBZ genes were analyzed in different tissues using eight phytohormones and four abiotic stress treatments based on RNA sequencing data and qRT-PCR verification. The results showed that each gene responded differently to more than one phytohormone or abiotic stress type. This research provides a foundation for future functional research on SlRBZ genes in tomato.

The role of altered translation in intellectual disability and epilepsy.

A very high proportion of cases of intellectual disability are genetic in origin and are associated with the occurrence of epileptic seizures during childhood. These two disorders together effect more than 5% of the world's population. One feature linking the two diseases is that learning and memory require the synthesis of new synaptic components and ion channels, while maintenance of overall excitability also requires synthesis of similar proteins in response to altered neuronal stimulation. Many of these disorders result from mutations in proteins that regulate mRNA processing, translation initiation, translation elongation, mRNA stability or upstream translation modulators. One theme that emerges on reviewing this field is that mutations in proteins that regulate changes in translation following neuronal stimulation are more likely to result in epilepsy with intellectual disability than general translation regulators with no known role in activity-dependent changes. This is consistent with the notion that activity-dependent translation in neurons differs from that in other cells types in that the changes in local cellular composition, morphology and connectivity that occur generally in response to stimuli are directly coupled to local synaptic activity and persist for months or years after the original stimulus.

Proteogenomics Integrating Reveal a Complex Network, Alternative Splicing, Hub Genes Regulating Heart Maturation.

Heart maturation is an essentially biological process for neonatal heart transition to adult heart, thus illustrating the mechanism of heart maturation may be helpful to explore postnatal heart development and cardiac cardiomyopathy. This study combined proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) and transcriptome analysis based on RNA sequencing to detect the proteins and genes associated with heart maturation in mice. The proteogenomics integrating analysis identified 254 genes/proteins as commonly differentially expressed between neonatal and adult hearts. Functional and pathway analysis demonstrated that these identified genes/proteins contribute to heart maturation mainly by regulating mRNA processing and energy metabolism. Genome-wide alternative splicing (AS) analysis showed that some important sarcomere and energy-associated genes undergo different AS events. Through the Cytoscape plug-in CytoHubba, a total of 23 hub genes were found and further confirmed by RT-qPCR. Next, we verified that the most up-regulated hub gene, Ogdhl, plays an essential role in heart maturation by detecting energy metabolism phenotype changes in the Ogdhl-interfering cardiomyocytes. Together, we revealed a complex gene network, AS genes and patterns, and candidate hub genes controlling heart maturation by proteome and transcriptome combination analysis.

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs.

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.

Survivin Expression Is Differentially Regulated by a Selective Cross-talk between RBM38 and miRNAs let-7b or miR-203a.

RNA-binding motif 38 (RBM38) is a member of a protein family with a highly conserved RNA-binding motif and has been shown to regulate mRNA processing, stability, and translation. Survivin is an essential modulator of apoptotic and nonapoptotic cell death as well as a stress responder. Survivin mRNA is the fourth most frequently overexpressed transcript in the human cancer transcriptome, and its aberrant expression is associated with chemo-/radioresistance and poor prognosis. In this study, we examined whether survivin expression is regulated by RBM38. RBM38 bound to survivin 3'-untranslated region and suppressed miRNA let-7b from binding to and degrading survivin mRNA, leading to increased survivin expression. RBM38 interacted with argonaute-2 (AGO2) and facilitated miR-203a-mediated degradation of survivin mRNA, leading to decreased survivin expression. Due to the abundance of let-7b over miR-203a, RBM38 ultimately increased survivin expression in HCT116 and MCF7 cells. In addition, Ser-195 in RBM38 interacted with Glu-73/-76 in AGO2, and Pep8, an eight-amino acid peptide spanning the region of Ser-195 in RBM38, blocked the RBM38-AGO2 interaction and inhibited miR-203a-mediated mRNA degradation, leading to enhanced survivin expression. Furthermore, Pep8 cooperated with YM155, an inhibitor of survivin, to suppress tumor spheroid growth and viability. Pep8 sensitized tumor cells to YM155-induced DNA damage in an RBM38-dependent manner. Together, our data indicate that RBM38 is a dual regulator of survivin and that Pep8/YM155 may be therapeutically explored for tumor suppression. SIGNIFICANCE: These findings show that RBM38 exerts opposing effects on survivin expression via two miRNAs, and disruption of the RBM38-AGO2 complex by an eight-amino acid peptide sensitizes tumor spheroids to survivin inhibitor YM155.

In vivo nuclear RNA structurome reveals RNA-structure regulation of mRNA processing in plants

BackgroundmRNA processing is critical for gene expression. A challenge in regulating mRNA processing is how to recognize the actual mRNA processing sites, such as splice and polyadenylation sites, when the sequence content is insufficient for this purpose. Previous studies suggested that RNA structure affects mRNA processing. However, the regulatory role of RNA structure in mRNA processing remains unclear.ResultsHere, we perform in vivo selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical profiling on Arabidopsis and generate the in vivo nuclear RNA structure landscape. We find that nuclear mRNAs fold differently from cytosolic mRNAs across translation start and stop sites. Notably, we discover a two-nucleotide single-stranded RNA structure feature upstream of 5′ splice sites that is strongly associated with splicing and the selection of alternative 5′ splice sites. The regulatory role of this RNA structure feature is further confirmed by experimental validation. Moreover, we find the single-strandedness of branch sites is also associated with 3′ splice site recognition. We also identify an RNA structure feature comprising two close-by single-stranded regions that is specifically associated with both polyadenylation and alternative polyadenylation events.ConclusionsWe successfully identify pre-mRNA structure features associated with splicing and polyadenylation at whole-genome scale and validate an RNA structure feature which can regulate splicing. Our study unveils a new RNA structure regulatory mechanism for mRNA processing.

Dynamic Patterns of N6-Methyladenosine Profiles of Messenger RNA Correlated with the Cardiomyocyte Regenerability during the Early Heart Development in Mice.

N6-Methyladenosine (m6A) plays important roles in regulating mRNA processing. Despite rapid progress in this field, little is known about the role and mechanism of m6A modification in myocardial development and cardiomyocyte regeneration. Existing studies have shown that the heart tissues of newborn mice have the capability of proliferation and regeneration, but its mechanism, particularly its relation to m6A methylation, remains unknown. Methods. To systematically profile the mRNA m6A modification pattern in the heart tissues of mice at different developmental stages, we jointly performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) of heart tissues of mice, respectively, aged 1 day old, 7 days old, and 28 days old. Results. We identified the linkages and association between differentially expressed mRNA transcripts and hyper or hypomethylated m6A peaks in C57BL/6J mice at different heart developmental stages. Results showed that the amount of m6A peaks and the level of m6A modification were the lowest in the heart of mice at 1 day old. By contrast, heart tissues from 7-day-old mice tended to possess the most m6A peaks and the highest global m6A level. However, the m6A characteristics of myocardial tissue changed little after 7 days old as compared to that of 1 day old. Specifically, we found 1269 downmethylated genes of 1434 methylated genes in 7-day-old mouse heart tissues as compared to those in 1-day-old mice. Hypermethylation of some specific genes may correlate with the heart's strong proliferative and regenerative capability at the first day after birth. In terms of m6A density, the tendency shifted from coding sequences (CDS) to 3′-untranslated regions (3′UTR) and stop codon with the progression of heart development. In addition, some genes demonstrated remarkable changes both in methylation and expression, like kiss1, plekha6, and megf6, which may play important roles in proliferation. Furthermore, signaling pathways highly related to proliferation such as “Wnt signaling pathway,” “ECM-receptor interaction,” and “cardiac chamber formation” were significantly enriched in 1-day-old methylated genes. Conclusions. Our results reveal a pattern that different m6A modifications are distributed in C57BL/6J heart tissue at different developmental stages, which provides new insights into a novel function of m6A methylation of mRNA in myocardial development and regeneration.

Distinct p63 and p73 Protein Interactions Predict Specific Functions in mRNA Splicing and Polyploidy Control in Epithelia.

Epithelial organs are the first barrier against microorganisms and genotoxic stress, in which the p53 family members p63 and p73 have both overlapping and distinct functions. Intriguingly, p73 displays a very specific localization to basal epithelial cells in human tissues, while p63 is expressed in both basal and differentiated cells. Here, we analyse systematically the literature describing p63 and p73 protein–protein interactions to reveal distinct functions underlying the aforementioned distribution. We have found that p73 and p63 cooperate in the genome stability surveillance in proliferating cells; p73 specific interactors contribute to the transcriptional repression, anaphase promoting complex and spindle assembly checkpoint, whereas p63 specific interactors play roles in the regulation of mRNA processing and splicing in both proliferating and differentiated cells. Our analysis reveals the diversification of the RNA and DNA specific functions within the p53 family.

Cytoskeletal disorganization underlies PABPN1-mediated myogenic disability

Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy.

PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation.

Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3' UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate5%), respectively. The majority of the peaks were preferentially located around the 3' UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels.

Abstract 4827: N6-Methyladenosine modification of LINC00901 promotes pancreatic cancer progression

Abstract N6-methyladenosine (m6A) is one of the most prevalent modifications of mRNAs in eukaryotes. Numerous recent studies have indicated that m6A plays the critical role in multiple physiologic functions and disease processes by regulation of mRNA processing, splicing, translation, and stability. Although the majority of RNA methylation studies focus on mRNAs, little is known whether long-coding RNAs (lncRNAs) are also subject to m6A modification. Therefore, this study aims to identify m6A modified lncRNAs and their functional role in pancreatic cancer. We performed methylated RNA immunoprecipitation (MeRIP) profiling against a focused group of lncRNAs and identified five lncRNA candidates, among which the highly enriched lncRNA was LINC00901. RNA dot blot, m6A RNA methylation quantification detection and in vivo RNA pulldown combined with m6A RNA methylation Western Blot confirmed the m6A modification of LINC00901. Of note, the analysis of TCGA datasets indicated that the high level of LINC00901 is associated with the poor overall survival (OS) of pancreatic ductal adenocarcinoma (PDAC) patients. Consistent with this result, we showed that LINC00901 promotes pancreatic cancer cell growth. Importantly, we identified that YTHDF1 serves as a “reader” of the m6A modified LINC00901 and furthermore, ectopic expression of YTHDF1 causes downregulation of LINC00901, suggesting a role for m6A modification in regulation of LINC00901 expression. Experiments are underway to determine how LINC00901 promotes pancreatic cancer cell growth. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. N6-Methyladenosine modification of LINC00901 promotes pancreatic cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4827.

Limited Proteolysis of Cyclooxygenase-2 Enhances Cell Proliferation.

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (ΔNT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the ΔNT- COX-2 protein also interacts in the nucleus with β-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis.

Cold-inducible RNA-binding protein Cirp, but not Rbm3, may regulate transcript processing and protection in tissues of the hibernating ground squirrel.

RNA-binding proteins (RBPs) have important roles in transcription, pre-mRNA processing/transport, mRNA degradation, translation, and non-coding RNA processing, among others. RBPs that are expressed in response to cold stress, such as Cirp and Rbm3, could regulate RNA stability and translation in hibernating mammals that reduce their body temperatures from 37°C to as low as 0-5°C during torpor bouts. RBPs including Cirp, Rbm3, and stress-inducible HuR translocate from the nucleus to stabilize mRNAs in the cytoplasm, and thereby could regulate which mRNA transcripts are protected from degradation and are translated, versus stored, for future protein synthesis or degraded by nucleases during cell stress associated with metabolic rate depression. This is the first study to explore the transcriptional/translational regulation, and subcellular localization of cold-inducible RBPs in a model hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus). Cirp protein levels were upregulated in liver, skeletal muscle, and brown adipose tissue throughout the torpor-arousal cycle whereas Rbm3 protein levels stayed constant or decreased, suggesting an important role for Cirp, but likely not Rbm3, in the hibernator stress response. Increased cytoplasmic localization of Cirp in liver and muscle and HuR in liver during torpor, but no changes in the relative levels of Rbm3 in the cytoplasm, emphasizes a role for Cirp and possibly HuR in regulating mRNA processing during torpor. This study informs our understanding of the natural adaptations that extreme animals use in the face of stress, and highlight natural stress response mediators that could be used to bolster cryoprotection of human organs donated for transplant.

G-quadruplexes in mRNA: A key structure for biological function

The last several years have seen exciting advances in the understanding of the structure and function of higher-order structures of RNA. Expression levels of some specific genes were shown to be directly regulated by environmentally-responsive formation of certain secondary structures such as stem-loops and pseudoknots. Even among these noncanonical structures, RNA G-quadruplexes, which form on the regions of guanine-rich sequences in mRNA, are highly stable structures that are involved in a variety of biological processes. However, many questions regarding the biological significance of RNA G-quadruplexes remain unsettled, mainly because it is difficult to locate the structures in mRNA. This review focuses on emerging methods that locate RNA G-quadruplexes in mRNA by computational and biochemical techniques. In addition, recent reports on the biological functions of RNA G-quadruplexes are also covered to highlight their various roles in cells, such as in regulating mRNA processing and translation.

Comparative Gene Expression Profiles in Parathyroid Adenoma and Normal Parathyroid Tissue.

Parathyroid adenoma is the main cause of primary hyperparathyroidism, which is characterized by enlarged parathyroid glands and excessive parathyroid hormone secretion. Here, we performed transcriptome analysis, comparing parathyroid adenomas with normal parathyroid gland tissue. RNA extracted from ten parathyroid adenoma and five normal parathyroid samples was sequenced, and differentially expressed genes (DEGs) were identified using strict cut-off criteria. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using DEGs as the input, and protein-protein interaction (PPI) networks were constructed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and visualized in Cytoscape. Among DEGs identified in parathyroid adenomas (n = 247; 45 up-regulated, 202 down-regulated), the top five GO terms for up-regulated genes were nucleoplasm, nucleus, transcription DNA-template, regulation of mRNA processing, and nucleic acid binding, while those for down-regulated genes were extracellular exosome, membrane endoplasmic reticulum (ER), membrane, ER, and melanosome. KEGG enrichment analysis revealed significant enrichment of five pathways: protein processing in ER, protein export, RNA transport, glycosylphosphatidylinositol-anchor biosynthesis, and pyrimidine metabolism. Further, PPI network analysis identified a densely connected sub-module, comprising eight hub molecules: SPCS2, RPL23, RPL26, RPN1, SEC11C, SEC11A, RPS25, and SEC61G. These findings may be helpful in further analysis of the mechanisms underlying parathyroid adenoma development.

RNA-Binding Proteins in the Control of LPS-Induced Macrophage Response.

Innate immune response is triggered by pathogen components, like lipopolysaccharides (LPS) of gram-negative bacteria. LPS initiates Toll-like receptor 4 (TLR4) signaling, which involves mitogen activated protein kinases (MAPK) and nuclear factor kappa B (NFκB) in different pathway branches and ultimately induces inflammatory cytokine and chemokine expression, macrophage migration and phagocytosis. Timely gene transcription and post-transcriptional control of gene expression confer the adequate synthesis of signaling molecules. As trans-acting factors RNA binding proteins (RBPs) contribute significantly to the surveillance of gene expression. RBPs are involved in the regulation of mRNA processing, localization, stability and translation. Thereby they enable rapid cellular responses to inflammatory mediators and facilitate a coordinated systemic immune response. Specific RBP binding to conserved sequence motifs in their target mRNAs is mediated by RNA binding domains, like Zink-finger domains, RNA recognition motifs (RRM), and hnRNP K homology domains (KH), often arranged in modular arrays. In this review, we focus on RBPs Tristetraprolin (TTP), human antigen R (HUR), T-cell intracellular antigen 1 related protein (TIAR), and heterogeneous ribonuclear protein K (hnRNP K) in LPS induced macrophages as primary responding immune cells. We discuss recent experiments employing RNA immunoprecipitation and microarray analysis (RIP-Chip) and newly developed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), photoactivatable ribonucleoside-enhanced crosslinking (PAR-iCLIP) and RNA sequencing techniques (RNA-Seq). The global mRNA interaction profile analysis of TTP, HUR, TIAR, and hnRNP K exhibited valuable information about the post-transcriptional control of inflammation related gene expression with a broad impact on intracellular signaling and temporal cytokine expression.