scholarly journals Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2459
Author(s):  
Olga Patutina ◽  
Daria Chiglintseva ◽  
Elena Bichenkova ◽  
Svetlana Gaponova ◽  
Nadezhda Mironova ◽  
...  
Keyword(s):  
Mirna Target ◽  
Catalytic Performance ◽  
Rnase H ◽  
Therapeutic Interventions ◽  
Mirna Sequence ◽  
Distinct Advantage ◽  
Rna Sequences ◽  
Ph Optimum ◽  
Additional Advantage ◽  
Recognition Motifs

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses–miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide “shoulders” of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5–7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5′- and 3′-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.

scholarly journals Transcriptome-wide analysis of PGC-1α–binding RNAs identifies genes linked to glucagon metabolic action

2020 ◽  
Vol 117 (36) ◽  
pp. 22204-22213 ◽  
Author(s):  
Clint D. J. Tavares ◽  
Stefan Aigner ◽  
Kfir Sharabi ◽  
Shashank Sathe ◽  
Beste Mutlu ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Large Fraction ◽  
Glucose Production ◽  
Peroxisome Proliferator ◽  
Rna Sequences ◽  
Peroxisome Proliferator Activated Receptor ◽  
Rna Recognition Motifs ◽  
Transcript Levels ◽  
Recognition Motifs ◽  
Metabolic Action

The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a transcriptional coactivator that controls expression of metabolic/energetic genes, programming cellular responses to nutrient and environmental adaptations such as fasting, cold, or exercise. Unlike other coactivators, PGC-1α contains protein domains involved in RNA regulation such as serine/arginine (SR) and RNA recognition motifs (RRMs). However, the RNA targets of PGC-1α and how they pertain to metabolism are unknown. To address this, we performed enhanced ultraviolet (UV) cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq) in primary hepatocytes induced with glucagon. A large fraction of RNAs bound to PGC-1α were intronic sequences of genes involved in transcriptional, signaling, or metabolic function linked to glucagon and fasting responses, but were not the canonical direct transcriptional PGC-1α targets such as OXPHOS or gluconeogenic genes. Among the top-scoring RNA sequences bound to PGC-1α wereFoxo1,Camk1δ,Per1,Klf15,Pln4,Cluh,Trpc5,Gfra1, andSlc25a25. PGC-1α depletion decreased a fraction of these glucagon-induced messenger RNA (mRNA) transcript levels. Importantly, knockdown of several of these genes affected glucagon-dependent glucose production, a PGC-1α–regulated metabolic pathway. These studies show that PGC-1α binds to intronic RNA sequences, some of them controlling transcript levels associated with glucagon action.

scholarly journals U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

1989 ◽  
Vol 9 (8) ◽  
pp. 3350-3359 ◽  
Author(s):  
D L Black ◽  
A L Pinto
Keyword(s):  
Rna Structure ◽  
Rna Secondary Structure ◽  
Rnase H ◽  
Rna Sequences ◽  
Large Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
The Individual ◽  
Nuclear Ribonucleoprotein ◽  
Dependent Interaction

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

scholarly journals A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

1997 ◽  
Vol 17 (5) ◽  
pp. 2649-2657 ◽  
Author(s):  
H Shi ◽  
B E Hoffman ◽  
J T Lis
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Loop Structure ◽  
Hairpin Loop ◽  
Structural Element ◽  
Rna Sequences ◽  
Sr Protein ◽  
Rna Recognition Motifs ◽  
Splicing Regulators ◽  
Recognition Motifs

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

scholarly journals Role of MiRNAs and It’s Single Nucleotide Polymorphism in Breast Cancer

2021 ◽  
Author(s):  
Debarshi Roy ◽  
Ramesh Bandla ◽  
Praveen Boddana ◽  
Rajesh Medisetty ◽  
Raghu Gogada
Keyword(s):  
Breast Cancer ◽  
Mirna Expression ◽  
Diagnosis And Treatment ◽  
Mirna Sequence ◽  
Diagnostic Tools ◽  
Rna Sequences ◽  
Ki 67 ◽  
Single Nucleotide ◽  
Cancer Occurrence ◽  
Non Coding Rna

MiRNAs are 20-22 nucleotide long single-stranded non-coding RNA sequences, which can regulate post transcriptional activity of mRNA by binding with it at 3’UTR region (untranslated region). Thus deregulation of miRNA expression is responsible for dysregulating mRNA function which contributes in developing various diseases as well as cancerous phenotypes. Alteration of single nucleotide in miRNA sequence is one of the reasons behind deregulation of miRNA expression. The most frequent carcinoma in current day is breast cancer which causes a high mortality among women around the world as well as India. Despite of the advancement of diagnostic tools, strategies and treatment, the cases of breast cancer is increasing every year. There are plenty of biomarkers like ER, PR, Her2, Ki-67, etc available which are frequently used in diagnosis and treatment of breast cancer. After the discovery of MiRNA in 1993 in Caenorhabiditis elegans, it is attracting all the limelight in diagnosis and treatment of different carcinomas as well as breast cancer. In this review we will discuss on involvement of different types of MiRNAs and miR SNPs in breast cancer occurrence and susceptibility in a detailed manner.

scholarly journals A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

2020 ◽  
Vol 48 (6) ◽  
pp. 2897-2911 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Giovanni L Burke ◽  
Ziyan Chen ◽  
Jack F Greenblatt ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Nascent Transcripts ◽  
Target Rna

Abstract The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

scholarly journals Dissecting the DNA binding landscape and gene regulatory network of p63 and p53

2020 ◽  
Author(s):  
Konstantin Riege ◽  
Helene Kretzmer ◽  
Arne Sahm ◽  
Simon S. McDade ◽  
Steve Hoffmann ◽  
...  
Keyword(s):  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Target Genes ◽  
De Novo ◽  
Therapeutic Interventions ◽  
Expression Studies ◽  
Gene Regulatory ◽  
Recognition Motifs ◽  
Gene Expression Studies ◽  
Search Approach

AbstractThe transcription factor (TF) p53 is the best-known tumor suppressor, but its ancient sibling p63 (ΔNp63) is a master regulator of epidermis development and a key oncogenic driver in squamous cell carcinomas (SCC). Despite multiple gene expression studies becoming available in recent years, the limited overlap of reported p63-dependent genes has made it difficult to decipher the p63 gene regulatory network (GRN). In particular, analyses of p63 response elements differed substantially among the studies. To address this intricate data situation, we provide an integrated resource that enables assessing the p63-dependent regulation of any human gene of interest. Here, we use a novel iterative de novo motif search approach in conjunction with extensive publicly available ChIP-seq data to achieve a precise global distinction between p53 and p63 binding sites, recognition motifs, and potential co-factors. We integrate all these data with enhancer:gene associations to predict p63 target genes and identify those that are commonly de-regulated in SCC and, thus, may represent candidates for therapeutic interventions.

scholarly journals Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1732
Author(s):  
Yaroslav Staroseletz ◽  
Svetlana Gaponova ◽  
Olga Patutina ◽  
Elena Bichenkova ◽  
Bahareh Amirloo ◽  
...  
Keyword(s):  
Metal Ion ◽  
Protein Complexes ◽  
Artificial Ribonucleases ◽  
Rna Sequences ◽  
Molecular Scaffold ◽  
Catalytically Active ◽  
Recognition Motifs ◽  
The One ◽  
Site Selective

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

scholarly journals An essential RNA-binding lysine residue in the Nab3 RRM domain undergoes mono and trimethylation

2019 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Kyle Biggar ◽  
Marc D. Meneghini
Keyword(s):  
Rna Binding ◽  
Growth Defect ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Target Rna

AbstractThe Nrd1-Nab3-Sen1 (NNS) complex integrates molecular inputs to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by methylation of histone H3 lysine-4 as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3, and Sen1 that are mono-, di-, or trimethylated, suggesting novel molecular inputs for NNS regulation. One of these methylated residues, Nab3 lysine-363 (K363), resides within its RRM, and is known to physically contact target RNA. Although mutation of Nab3-K363 to arginine (Nab3-K363R) causes a severe growth defect, it nevertheless produces a stable protein that is incorporated into the NNS complex, suggesting that RNA binding through Nab3-K363 is crucial for NNS function. Consistent with this hypothesis, K363R mutation decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects in vivo. These findings reveal crucial roles for Nab3-K363 and suggest that methylation of this residue may modulate NNS activity through its impact on Nab3 RNA binding.

scholarly journals Structural differences between pri-miRNA paralogs promotes alternative Drosha cleavage and expands target repertoires

2018 ◽  
Author(s):  
Xavier Bofill-De Ros ◽  
Wojciech K. Kasprzak ◽  
Yuba Bhandari ◽  
Lixin Fan ◽  
Quinn Cavanaugh ◽  
...  
Keyword(s):  
Mirna Target ◽  
Downstream Processing ◽  
Mirna Sequence ◽  
Low Grade ◽  
Seed Sequence ◽  
Glioma Patient ◽  
Structural Differences ◽  
Flexible Stem ◽  
Stem Structure ◽  
Distinct Role

AbstractMicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low grade glioma patient samples indicate that the alternative-miR-9 plays a distinct role in preventing tumor progression. To generalize our model, we provide evidence that distortion of pri-miRNA stems correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.

scholarly journals Dissecting the DNA binding landscape and gene regulatory network of p63 and p53

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Konstantin Riege ◽  
Helene Kretzmer ◽  
Arne Sahm ◽  
Simon S McDade ◽  
Steve Hoffmann ◽  
...  
Keyword(s):  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Target Genes ◽  
De Novo ◽  
Therapeutic Interventions ◽  
Gene Regulatory ◽  
Recognition Motifs ◽  
Gene Expression Studies ◽  
Search Approach ◽  
Transcription Factor P53

The transcription factor p53 is the best-known tumor suppressor, but its sibling p63 is a master regulator of epidermis development and a key oncogenic driver in squamous cell carcinomas (SCC). Despite multiple gene expression studies becoming available, the limited overlap of reported p63-dependent genes has made it difficult to decipher the p63 gene regulatory network. Particularly, analyses of p63 response elements differed substantially among the studies. To address this intricate data situation, we provide an integrated resource that enables assessing the p63-dependent regulation of any human gene of interest. We use a novel iterative de novo motif search approach in conjunction with extensive ChIP-seq data to achieve a precise global distinction between p53-and p63-binding sites, recognition motifs, and potential co-factors. We integrate these data with enhancer:gene associations to predict p63 target genes and identify those that are commonly de-regulated in SCC representing candidates for prognosis and therapeutic interventions.

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