RESUMO
Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.
Assuntos
Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação Eucariótico 4G/metabolismo , Neoplasias Hematológicas/metabolismo , Peptídeos/química , Biossíntese de Proteínas , Animais , Progressão da Doença , Genoma Humano , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fases de Leitura Aberta , Polirribossomos/química , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Ribossomos/metabolismo , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.
Assuntos
Archaea/genética , Interação Gene-Ambiente , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Archaea/metabolismo , Regulação da Expressão Gênica em Archaea , RNA Arqueal/metabolismo , Transdução de Sinais/genéticaRESUMO
Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, the in vitro preparation of dsRNA is costly and laborious. In this study, we have developed a novel and interesting method designated as pfRCT (promoter-free rolling-circle transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without directional preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced into the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just-transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of the dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.
Assuntos
RNA de Cadeia Dupla , Ribonuclease H , Ribonuclease H/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Transcrição GênicaRESUMO
Murine gammaherpesvirus 68 (MHV68) establishes latency mainly in B cells and causes lymphomas reminiscent of human gammaherpesvirus diseases in laboratory mice. To study the molecular mechanism of virus infection and how the viral determinants control cell and eventually cause tumorigenesis, readily available latently infected cell lines are essential. For in vitro MHV68 latency studies, only two cell culture systems have been available. Gammaherpesviruses are known to infect developing B cells and macrophages, therefore we aimed to expand the MHV68 latently infected cell line repertoire. Here, several latently infected immature B cell and macrophage-like cell line clones were generated. Hygromycin-resistant recombinant MHV68 was isolated from a laboratory-made latent cell line, HE2.1, and propagated to develop stable cell lines that carry the viral genome under hygromycin selection. Subclones of these cells lines were analyzed for viral miRNA expression by TaqMan qPCR and assessed for expression of a lytic viral transcript M3. The cell lines maintain the viral genome as an episome shown by the digestion-circularization PCR assay. Latently infected cell lines generated here do not express viral miRNAs higher than the parental cell line. However, these cell lines may provide an alternative tool to study latency mechanisms and miRNA target identification studies.
Assuntos
Genoma Viral , Higromicina B , Macrófagos , MicroRNAs , RNA Viral , Rhadinovirus , Latência Viral , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Latência Viral/genética , Higromicina B/farmacologia , Higromicina B/análogos & derivados , Macrófagos/virologia , Macrófagos/metabolismo , Rhadinovirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Linhagem Celular , Regulação Viral da Expressão Gênica , Células Precursoras de Linfócitos B/virologia , Células Precursoras de Linfócitos B/metabolismo , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , CinamatosRESUMO
Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3'-5' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5' end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3'-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3' extension of pre-16S rRNA, and so generated the mature 3' end. Circularized RT-PCR (cRT-PCR) and 5'-RACE identified two cleavage sites near helix 1 at the 5' end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species.
Assuntos
Mathanococcus , RNA Ribossômico 23S , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Mathanococcus/genética , RNA Ribossômico 5S , Archaea , RibonucleasesRESUMO
With the development of high-throughput sequencing, circular RNA has come into people's vision and attracted more and more attention. Many studies have found that circular RNA plays an important role in a variety of biological processes and the occurrence and development of diseases. According to the previous sequencing results, circRNA_3238 was differentially expressed in ALV-J infected group and the non-infected group was selected for subsequent verification and analysis. We found that circRNA_3238 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, lymph, and bursa of fabricius. Bioinformatics results show that circRNA_3238 and the predicted target genes enriched MAPK signaling pathway, Notch signaling pathway, and other pathways related to disease or immune, revealing circRNA_3238 may indirectly regulate the process of ALV-J infection by regulating target genes.
Assuntos
MicroRNAs , RNA Circular , Animais , RNA Circular/genética , RNA Circular/metabolismo , RNA/genética , Galinhas/genética , Galinhas/metabolismo , Transdução de Sinais/genética , Sistema de Sinalização das MAP Quinases , MicroRNAs/genéticaRESUMO
BACKGROUND & AIMS: To clarify the biological roles, circularization process and secretion pathway of circRHOBTB3 in colorectal cancer (CRC) progression. METHODS: We performed a comprehensive analysis of circRNA levels in serum exosomes from multiple types of cancer patients in public databases and verified the higher level of circRHOBTB3 in CRC sera versus healthy donors by RT-qPCR. Then, the function of circRHOBTB3 in CRC was investigated in vitro and in vivo. RNA-seq and RNA pull-down assays together with mass spectrometry identified the downstream signals and the binding proteins of circRHOBTB3. Finally, Antisense oligonucleotides (ASOs) were designed to target circularization and secretion elements of circRHOBTB3 for CRC therapy. RESULTS: circRHOBTB3 levels were increased in the sera but was downregulated in tissue samples in CRC, and the downregulation was associated with poor prognosis. Furthermore, circRHOBTB3 acts a tumor-suppressive circRNA by repressing metabolic pathways, intracellular ROS production in CRC. Several key elements were discovered to regulate circRHOBTB3 circularization and exosomal secretion. Moreover, SNF8 was identified that sorts circRHOBTB3 into exosomes. Interestingly, we found that CRC cells could actively secrete more circRHOBTB3 than normal cells. According to the sequence of regulatory elements for circularization and exosomal secretion, we designed and synthesized ASOs, which increased circRHOBTB3 expression and blocked circRHOBTB3 exosomal secretion. More importantly, ASOs could inhibit CRC growth and metastasis in vitro and in vivo. CONCLUSIONS: circRHOBTB3 plays a tumor-suppressive role in CRC and has to be excreted out of cells to sustain cancer cell fitness. ASOs targeting regulatory elements for circularization and exosomal secretion will become a novel antitumor strategy.
Assuntos
Neoplasias Colorretais , Exossomos , MicroRNAs , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Exossomos/metabolismo , Humanos , MicroRNAs/genética , RNA Circular/genéticaRESUMO
Circular RNA (circRNA) is a new non-coding RNA with a highly conserved and stable covalently closed loop structure, and it plays an important role in a variety of biological processes and the occurrence of diseases. Based on the sequencing results, circRNA_3079 had the most significant difference between the infected group and normal group, up to about 8 times. It has attracted our attention and was selected for further verification and analysis. Though the characteristics analysis of circRNA_3079 in chicken, we found circRNA_3079 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, spleen, lymph and bursa of fabricius. Bioinformatics analysis results showed that circRNA_3079 and the predicted target genes are enriched in many pathways related to immunity or tumors, such as p53 signaling pathway, Jak-STAT signaling pathway and NOD-like receptor signaling pathway, which revealed that circRNA_3079 may indirectly regulate the ALV-J infection process through the regulation of target genes.HIGHLIGHTSCircRNA_3079 is an abundant and stable circular RNA expressed in different tissues and cells in chicken.The circularization of circRNA_3079 does not depend on the reverse complementary repetitive sequence of the flanking sequence.CircRNA_3079 may indirectly regulate the ALV-J infection process.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Animais , Leucose Aviária/genética , Vírus da Leucose Aviária/genética , Galinhas/genética , Proteínas NLR , RNA Circular/genética , RNA não Traduzido , Proteína Supressora de Tumor p53RESUMO
mRNA is gaining success as a new therapeutic agent and vaccine. However, mRNA has limitations in stability. To overcome the shortcomings of mRNA, circular RNA is emerging as a new modality. In this review, several current methods of manufacturing circular RNA in vitro are introduced and their advantages and disadvantages are reviewed. Furthermore, this study discusses which fields and directions of research and development are needed for the increase in the efficacy and productivity of circular RNA as a therapeutic agent and vaccine formulation.
Assuntos
RNA Circular , RNA , RNA Mensageiro/genética , Técnicas In Vitro , RNA/genéticaRESUMO
Transposable elements (TEs) and their reverse complementary sequence pairs (RCPs) are enriched around loci that produce circular RNAs (circRNAs) in plants. However, the function of these TE-RCP pairs in modulating circRNA expression remains elusive. Here, we identified 4609 circRNAs in poplar (Populus tomentosa) and showed that miniature inverted repeat transposable elements (MITEs)-RCPs were enriched in circRNA flanking regions. Moreover, we used expression quantitative trait nucleotide (eQTN) mapping to decipher the cis-regulatory role of MITEs. eQTN results showed that 14 single-nucleotide polymorphisms (SNPs) were significantly associated with Circ_0000408 and Circ_0003418 levels and the lead associated SNPs were located in MITE-RCP regions, indicating that MITE-RCP sequence variations affect exon circularization. Overexpression and knockdown analysis showed that Circ_0003418 positively modulated its parental gene, which encodes the RING-type E3 ligase XBAT32, and specifically increased the expression of the PtoXBAT32.5 transcript variant, which lacks the E3 ubiquitin ligase domain. Under heat stress, PtoXBAT32.5 expression was induced with up-regulation of Circ_0003418, resulting in increased production of ethylene and peroxidation of membrane lipids. Our findings thus reveal the cis-regulatory mechanism by which a MITE-RCP pair affects circRNA abundance in poplar and indicate that Circ_0003418 is a negative regulator of poplar heat tolerance via the ubiquitin-mediated protein modification pathway.
Assuntos
Populus , Termotolerância , Elementos de DNA Transponíveis , Etilenos , Populus/genética , RNA CircularRESUMO
The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.
Assuntos
Regiões 5' não Traduzidas/genética , Proteínas do Capsídeo/fisiologia , Vírus da Dengue/metabolismo , Pareamento de Bases/genética , Sequência de Bases , Proteínas do Capsídeo/metabolismo , Sequência Conservada , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Genoma Viral/fisiologia , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA Circular/química , RNA Circular/genética , RNA Viral/química , RNA Viral/genética , Replicação Viral/genéticaRESUMO
RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.
Assuntos
Capsídeo/ultraestrutura , Vírus da Dengue/ultraestrutura , Genoma Viral , RNA Viral/ultraestrutura , Pareamento de Bases , Capsídeo/química , Capsídeo/metabolismo , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Aptidão Genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Sorogrupo , Montagem de Vírus/genéticaRESUMO
The ribosomal RNA (rRNA) genes are generally organized as an operon and cotranscribed into a polycistronic precursor; therefore, processing and maturation of pre-rRNAs are essential for ribosome biogenesis. However, rRNA maturation pathways of archaea, particularly of methanoarchaea, are scarcely known. Here, we thoroughly elucidated the maturation pathway of the rRNA operon (16S-tRNAAla-23S-tRNACys-5S) in Methanolobus psychrophilus, one representative of methanoarchaea. Enzymatic assay demonstrated that EndA, a tRNA splicing endoribonuclease, cleaved bulge-helix-bulge (BHB) motifs buried in the processing stems of pre-16S and pre-23S rRNAs. Northern blot and quantitative PCR detected splicing-coupled circularization of pre-16S and pre-23S rRNAs, which accounted for 2% and 12% of the corresponding rRNAs, respectively. Importantly, endoribonuclease Nob1 was determined to linearize circular pre-16S rRNA at the mature 3' end so to expose the anti-Shine-Dalgarno sequence, while circular pre-23S rRNA was linearized at the mature 5' end by an unknown endoribonuclease. The resultant 5' and 3' extension in linearized pre-16S and pre-23S rRNAs were finally matured through 5'-3' and 3'-5' exoribonucleolytic trimming, respectively. Additionally, a novel processing pathway of endoribonucleolysis coupled with exoribonucleolysis was identified for the pre-5S rRNA maturation in this methanogen, which could be also conserved in most methanogenic euryarchaea. Based on evaluating the phylogenetic conservation of the key elements that are involved in circularization and linearization of pre-rRNA maturation, we predict that the rRNA maturation mode revealed here could be prevalent among archaea.
Assuntos
Archaea/fisiologia , Regulação da Expressão Gênica em Archaea , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , Sequência de Bases , Sequência Conservada , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Modelos Biológicos , Conformação de Ácido Nucleico , Clivagem do RNA , Precursores de RNA/química , Splicing de RNA , RNA Ribossômico/química , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/genética , RNA de Transferência/genética , Motivos de Ligação ao RNARESUMO
It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m6A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5' end-independent translation initiation. Today a new family of so-called "noncoding" circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5' end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3'-5' interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.
Assuntos
Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , RNA Circular/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Proteínas de Ligação a Poli(A)/metabolismoRESUMO
Circular RNAs (CircRNAs) were first identified as a viroid and later found to also be an endogenous RNA splicing product in eukaryotes. In recent years, a series of RNA-sequencing analyses from a diverse range of eukaryotes have shed new light on these eukaryotic circRNAs, revealing dynamic expression patterns in various developmental stages and physiological conditions. In this review, we focus on circRNAs implicated in stress response pathways and explore potential mechanisms underlying their regulation. To date, circRNAs have been shown to act as scaffolds in the assembly of protein complexes, sequester proteins from native subcellular localization, activate transcription of parental genes, inhibit RNA-protein interactions, and function as regulators of microRNA activity. Although the mechanism modulating circRNA levels during stress remains unclear, circRNAs are shown to be regulated during biogenesis, degradation, and exportation. As circRNAs do not have 5' and 3' ends, there are no entry points for exoribonucleases to initiate degradation. Such inherent stability makes this class of RNA a strong candidate to maintain homeostasis in the face of environmental challenges.
Assuntos
RNA/metabolismo , Estresse Fisiológico , Animais , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , RNA/análise , RNA/genética , Estabilidade de RNA , RNA Circular , Transcrição GênicaRESUMO
BACKGROUND: Single-cell sequencing experiments use short DNA barcode 'tags' to identify reads that originate from the same cell. In order to recover single-cell information from such experiments, reads must be grouped based on their barcode tag, a crucial processing step that precedes other computations. However, this step can be difficult due to high rates of mismatch and deletion errors that can afflict barcodes. RESULTS: Here we present an approach to identify and error-correct barcodes by traversing the de Bruijn graph of circularized barcode k-mers. Our approach is based on the observation that circularizing a barcode sequence can yield error-free k-mers even when the size of k is large relative to the length of the barcode sequence, a regime which is typical single-cell barcoding applications. This allows for assignment of reads to consensus fingerprints constructed from k-mers. CONCLUSION: We show that for single-cell RNA-Seq circularization improves the recovery of accurate single-cell transcriptome estimates, especially when there are a high number of errors per read. This approach is robust to the type of error (mismatch, insertion, deletion), as well as to the relative abundances of the cells. Sircel, a software package that implements this approach is described and publically available.
Assuntos
DNA/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , HumanosRESUMO
Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 310 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
MAIN CONCLUSION: A total of 558 potential circular RNAs (circRNAs) were identified in citrus, and these were analyzed and compared. One hundred seventy-six differentially expressed circRNAs were identified in two genotypes of trifoliate orange. Circular RNAs (circRNAs) play diverse roles in transcriptional control and microRNA (miRNA) function. However, little information is known about circRNAs in citrus. To identify citrus circRNAs and investigate their functional roles, high-throughput sequencing of precocious trifoliate orange (an early-flowering trifoliate orange mutant, Poncirus trifoliata L. Raf.) and its wild type was performed. A total of 558 potential circRNAs were identified by bioinformatic analysis, and 86.02% of these were sense-overlapping circRNAs. Their sequence features, alternative circularization, and other characteristics were investigated in this study. Compared with the wild type, 176 circRNAs were identified as differentially expressed circRNAs, 61 were significantly up-regulated and 115 were down-regulated in precocious trifoliate orange, indicating that they may play an important role in the early flowering process. Alternative circularization and differential expression of some circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction. The functions of differentially expressed circRNAs and their host genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. We found that many differentially expressed circRNAs had abundant miRNA binding sites: 29 circRNAs were found to act as the 16 miRNA targets. Overall, these results will help to reveal the biological functions of circRNAs in growth and development of citrus.
Assuntos
Poncirus/genética , RNA de Plantas/genética , RNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genótipo , Poncirus/crescimento & desenvolvimento , RNA/fisiologia , RNA Circular , RNA de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.
Assuntos
DNA Circular/química , Eletroforese em Gel de Ágar/métodos , DNA Ligases/metabolismo , Fluorescência , Conformação de Ácido NucleicoRESUMO
In a recent issue of Nature Communications Ukleja and co-workers reported a cryo-EM 3D reconstruction of the Ccr4-Not complex from Schizosaccharomyces pombe with an immunolocalization of the different subunits. The newly gained architectural knowledge provides cues to apprehend the functional diversity of this major eukaryotic regulator. Indeed, in the cytoplasm alone, Ccr4-Not regulates translational repression, decapping and deadenylation, and the Not module additionally plays a positive role in translation. The spatial distribution of the subunits within the structure is compatible with a model proposing that the Ccr4-Not complex interacts with the 5' and 3' ends of target mRNAs, allowing different functional modules of the complex to act at different stages of the translation process, possibly within a circular constellation of the mRNA. This work opens new avenues, and reveals important gaps in our understanding regarding structure and mode of function of the Ccr4-Not complex that need to be addressed in the future.