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Background: Colorectal cancer (CRC) is one of the most common types of cancer worldwide. A number of dysregulated microRNAs (miRNAs) have been linked to CRC progression and treatment response and are thought to be promising prognostic biomarkers for this cancer. microRNA-330 (miR-330-5p) has been reported to inhibit cell proliferation through suppressing thymidylate synthase (TYMS). In the current study, miR-330-5p, TYMS, and their interactions were investigated to evaluate their therapeutic and diagnostic value for CRC treatment. Methods: The expression levels of miR-330-5p and TYMS were evaluated in silico using TCGA datasets for CRC. Data validation was performed on a set of internal samples (100 pairs of CRC tumor specimens and adjacent non-cancerous samples) utilizing real-time PCR assay. The linkage between clinicopathological parameters and expression levels was also investigated. Results: TCGA results indicated that miR-330-5p and TYMS were significantly upregulated and downregulated in the CRC, respectively. Real-time PCR results confirmed that the expression of miR-330-5p was significantly upregulated in tumor tissues relative to marginal tissues (P = 0.0005), whereas TYMS expression was significantly downregulated (P = 0.0001). The transcript level of miR-330-5p was associated with tumor stage and lymph node metastases. Conclusion: The microRNA-330 inhibited cell proliferation by suppressing thymidylate synthase (TYMS) in colorectal cancer. Therefore, suggesting that they are valuable factors for further studies of alternative treatment and diagnostic methods. © 2021, Springer Science+Business Media, LLC, part of Springer Nature., This study was supported by a grant from the research deputy of the Department of Tuberculosis and Lung Diseases Research Center, University Tabriz University of Medical Sciences, Tabriz, Iran.

Background: Aberrant expression of long noncoding RNAs (lncRNAs) is associated with the progression of human cancers, including gastric cancer (GC). The function of lncRNA DLGAP1-AS2, as an oncogene, has been identified in glioma, hepatocellular carcinoma, and cholangiocarcinoma but not in other malignancies. Therefore, this study was aimed to explore the association of DLGAP1-AS2 with gastric tumorigenesis and beyond.Methods and Results: The expression level of DLGAP1-AS2 was prevaluated in GC datasets from Gene Expression Omnibus (GEO). Moreover, qRT-PCR experiment was performed on 25 paired GC and adjacent normal tissue samples. The Cancer Genome Atlas (TCGA) data were also analyzed for further validations. Consistent with data obtained from GEO datasets, qRT-PCR results revealed that DLGAP1-AS2 was significantly (p < 0.0032) upregulated in GC specimens compared to normal samples, which was additionally confirmed using TCGA analysis (p). DLGAP1-AS2 expression level was also correlated with age (p =0.0008), lymphatic and vascular invasion (p =0.0415) in internal samples. Also, a significant correlation was found between DLGAP1-AS2 and YAP1 expression, as its valid downstream target, in GC samples. Besides, analysis of other prevalent tumor entities using TCGA illustrated the significant overexpression of DLGAP1-AS2 in lung, colorectal, and prostate cancers, further indicating its promise as an oncogene. Moreover, ROC curve analysis showed the high accuracy of the DLGAP1-AS2 expression pattern as a diagnostic biomarker for gastric and colorectal cancers. Conclusion: Our findings indicated that DLGAP1-AS2 might display oncogenic property in gastric tumorigenesis and be suggested as a therapeutic and diagnostic target.

Aim MYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. Materials and methods Cells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. Results Our results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. Conclusion We observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans-acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs. Fil:Nogués, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Cramer, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:De la Mata, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fededa, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Blaustein, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Srebrow, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Enhancing brown fat activity and promoting white fat browning are attractive therapeutic strategies for treating obesity and associated metabolic disorders. To provide a comprehensive picture of the gene regulatory network in these processes, we conducted a series of transcriptome studies by RNA sequencing (RNA-seq) and quantified the mRNA and long noncoding RNA (lncRNA) changes during white fat browning (chronic cold exposure, beta-adrenergic agonist treatment, and intense exercise) and brown fat activation or inactivation (acute cold exposure or thermoneutrality, respectively). mRNA–lncRNA coexpression networks revealed dynamically regulated lncRNAs to be largely embedded in nutrient and energy metabolism pathways. We identified a brown adipose tissue–enriched lncRNA, lncBATE10, that was governed by the cAMP-cAMP response element-binding protein (Creb) axis and required for a full brown fat differentiation and white fat browning program. Mechanistically, lncBATE10 can decoy Celf1 from Pgc1α, thereby protecting Pgc1α mRNA from repression by Celf1. Together, these studies provide a comprehensive data framework to interrogate the transcriptomic changes accompanying energy homeostasis transition in adipose tissue., Author summary Fat accumulation is a major health problem in many countries, but unlike white fat—which stores calories—brown fat is packed with mitochondria to burn energy. Therefore, promoting “browning” of white fat and enhancing brown fat activity are seen as promising therapeutic strategies to fight obesity. Long noncoding RNAs (lncRNAs), once largely believed to be functionally irrelevant, are receiving special attention because of the recent realization of their important role in many biological processes. Here, we performed a series of transcriptome analyses, including lncRNAs, during white fat browning and brown fat activation. Based on the mRNA–lncRNA coexpression network, we identified brown adipose tissue–enriched lncRNA 10 (lncBATE10) as a new regulator in brown adipocyte differentiation. Loss of lncBATE10 impaired expression of a brown fat–selective program in brown adipocytes and during browning of white adipocytes. We further showed that lncBATE10 could facilitate browning of white fat and brown fat activation by promoting Pgc1a expression. Taken together, we depicted a comprehensive noncoding transcriptome network during white fat browning and brown fat activation and identified lncBATE10 as a novel regulator in these processes.

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15 N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional ( 15 N, 1 H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and a-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.

The nucleolar proteins Gar1p and fibrillarin possess a typical nucleolar glycine/arginine-rich domain and belong to ribonucleoprotein particles. Both proteins are essential for yeast cell growth and are required for pre-rRNA processing. In addition, Gar1p is involved in pre-rRNA pseudouridylation, whereas fibrillarin is required for pre-rRNA methylation. Gar1p and fibrillarin are each associated with a different subset of the small nucleolar RNAs (snoRNAs). Gar1p is co-immunoprecipitated with the H/ACA family of snoRNAs, whereas fibrillarin is co-immunoprecipitated with the C/D family. However, attempts to demonstrate direct interactions between fibrillarin and snoRNAs have failed, and such interactions between Gar1p and the H/ACA snoRNAs had not get been reported. Among the H/ACA snoRNAs associated with Gar1p, one can distinguish a large group of snoRNAs that are not essential in yeast and serve as guides for pseudouridine synthesis onto the pre-rRNA molecule. In contrast, the two snoRNAs snR10 and snR30 are required for normal cell growth and for pre-rRNA cleavage. We show here that Gar1p interacts in vitro directly and specifically with these two snoRNAs, Deletion analysis of Gar1p indicates that a major RNA binding element, which is extremely well conserved throughout evolution, lies in the middle of the protein. However, this domain alone binds poorly to the target RNAs and an accessory domain is required to restore efficient binding. The accessory domain can be either one of the glycine/arginine-rich domains or a second element of the core of the protein that is highly conserved between different species. ispartof: Journal of biological chemistry vol:273 issue:18 pages:10868-10873 ispartof: location:United States status: published

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from â ̂1/410 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide. © 2014 Nature America, Inc.

MicroRNAs (miRNAs) are 20-22 nucleotides long small non-coding RNAs that regulate gene expression post-transcriptionally. Last decade has witnessed emerging evidences of active roles of miRNAs in tumor development, progression, metastasis, and drug resistance. Many factors contribute to their dysregulation in cancer, such as chromosomal aberrations, differential methylation of their own or host genes' promoters and alterations in miRNA biogenesis pathways. miRNAs have been shown to act as tumor suppressors or oncogenes depending on the targets they regulate and the tissue where they are expressed. Because miRNAs can regulate dozens of genes simultaneously and they can function as tumor suppressors or oncogenes, they have been proposed as promising targets for cancer therapy. In this review, we focus on the role of miRNAs in driving drug resistance and metastasis which are associated with stem cell properties of cancer cells. Furthermore, we discuss systems biology approaches to combine experimental and computational methods to study effects of miRNAs on gene or protein networks regulating these processes. Finally, we describe methods to target oncogenic or replace tumor suppressor miRNAs and current delivery strategies to sensitize refractory cells and to prevent metastasis. A holistic understanding of miRNAs' functions in drug resistance and metastasis, which are major causes of cancer-related deaths, and the development of novel strategies to target them efficiently will pave the way towards better translation of miRNAs into clinics and management of cancer therapy. © 2014 Springer-Verlag Berlin Heidelberg.

The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level. Refereed/Peer-reviewed

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ~20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. © 2012 Schor et al. Fil:Schor, I.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Risso, G.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

It has been well established that the elastic character of DNA plays an important role in protein-DNA binding specificity. In this work, we have analyzed the role of elasticity to understand the binding specificity of protein-RNA complexes. We have developed a sequence dependent stiffness scale for the trinucleotides in RNA and revealed the similarities and differences compared with DNA. We found that the stiffness of 15 trinucleotides has inverse effects and nine nucleotides are significantly different between RNA and DNA. The analysis on the relationship between RNA stiffness and RNA-binding specificity shows that the influence of elasticity is minimal in protein-RNA recognition, whereas it plays an important role in protein-DNA binding specificity. We observed a moderate correlation between stiffness and dissociation constant in U1A RBD1 protein and PP7 coat protein, whereas the correlation is poor for many other complexes. These results show that along with RNA stiffness, other interactions, such as shape complementarity, electrostatic interactions, hydrogen bonds and direct contacts between RNA and protein atoms are important for protein-RNA recognition. � 2012 Bentham Science Publishers.

cited By 46; International audience; In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5′-untranslated region (5′-UTR), including a stretch of pyrimidines at the 5′-end. The 5′-terminal oligopyrimidine (5′-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5′-UTR downstream from the 5′-TOP sequence.

FMR1 is an RNA-binding protein that is either absent or mutated in, patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an :N-terminal stretch of 200 amino acids, the two KH-regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular,dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N-and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(ra). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximate to 400 residues, whereas the C-terminus provides a nonspecific binding surface. ispartof: Rna-a publication of the rna society vol:5 issue:9 pages:1248-1258 ispartof: location:United States status: published