scholarly journals SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts

2019 ◽  
Author(s):  
Katharina Hutter ◽  
Michael Lohmüller ◽  
Almina Jukic ◽  
Felix Eichin ◽  
Seymen Avci ◽  
...  
Keyword(s):  
Noncoding Rnas ◽  
General Mechanism ◽  
List Type ◽  
Loop Structure ◽  
Stem Loop ◽  
Protein Coding ◽  
Mirna Processing ◽  
Small Noncoding Rnas ◽  
Stem Loop Structure ◽  
Attachment Factor

SummaryMicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally silence most protein-coding genes in mammals. They are generated from primary transcripts containing single or multiple clustered stem-loop structures that are thought to be recognized and cleaved by the DGCR8/DROSHA Microprocessor complex as independent units. Contrasting this view, we here report an unexpected mode of processing of a bicistronic cluster of the miR-15 family, miR-15a-16-1. We find that the primary miR-15a stem-loop is a poor Microprocessor substrate and is consequently not processed on its own, but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage, and describe SAFB2 as a novel accessory protein of DROSHA. Notably, SAFB2-mediated cluster assistance expands to other clustered pri-miRNAs including miR-15b, miR-92a and miR-181b, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal DGCR8/DROSHA substrates in clustered primary miRNA transcripts.Highlightsthe primary miR-15a stem-loop structure per se is a poor Microprocessor substratecleavage of pri-miR-15a requires the processing of an additional miRNA stem-loop on the same RNAsequential pri-miRNA processing or “cluster assistance” is mediated by SAFB proteinsSAFB2 associates with the Microprocessor

scholarly journals Rep-Mediated Nicking of the Adeno-Associated Virus Origin Requires Two Biochemical Activities, DNA Helicase Activity and Transesterification

1999 ◽  
Vol 73 (11) ◽  
pp. 9325-9336 ◽  
Author(s):  
J. Rodney Brister ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Helicase ◽  
Regulatory Function ◽  
General Mechanism ◽  
Loop Structure ◽  
Helicase Activity ◽  
Stem Loop ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Stem Loop Structure ◽  
Virus Origin

ABSTRACT The single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trssequence necessary for Rep cleavage, 3′-CCGGT/TG-5′, and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at thetrs. Interestingly, Rep nicking on a TR substrate that fixes this trs stem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trs and extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function.

scholarly journals Regulation of Histone H4 Lys16 Acetylation by Predicted Alternative Secondary Structures in roX Noncoding RNAs

2008 ◽  
Vol 28 (16) ◽  
pp. 4952-4962 ◽  
Author(s):  
Seung-Won Park ◽  
Mitzi I. Kuroda ◽  
Yongkyu Park
Keyword(s):  
Drosophila Melanogaster ◽  
X Chromosome ◽  
Noncoding Rnas ◽  
Loop Structure ◽  
Histone H4 ◽  
Stem Loop ◽  
Functional Conservation ◽  
Stem Loop Structure ◽  
Msl Complex ◽  
H4k16 Acetylation

ABSTRACT Despite differences in size and sequence, the two noncoding roX1 and roX2 RNAs are functionally redundant for dosage compensation of the Drosophila melanogaster male X chromosome. Consistent with functional conservation, we found that roX RNAs of distant Drosophila species could complement D. melanogaster roX mutants despite low homology. Deletion of a conserved predicted stem-loop structure in roX2, containing a short GUb (GUUNUACG box) in its 3′ stem, resulted in a defect in histone H4K16 acetylation on the X chromosome in spite of apparently normal localization of the MSL complex. Two copies of the GUb sequence, newly termed the “roX box,” were functionally redundant in roX2, as mutants in a single roX box had no phenotype, but double mutants showed reduced H4K16 acetylation. Interestingly, mutation of two of three roX boxes in the 3′ end of roX1 RNA also reduced H4K16 acetylation. Finally, fusion of roX1 sequences containing a roX box restored function to a roX2 deletion RNA lacking its cognate roX box. These results support a model in which the functional redundancy between roX1 and roX2 RNAs is based, at least in part, on short GUUNUACG sequences that regulate the activity of the MSL complex.

scholarly journals The Red Clover Necrotic Mosaic Virus RNA2 trans-Activator Is Also a cis-Acting RNA2 Replication Element

2005 ◽  
Vol 79 (2) ◽  
pp. 978-986 ◽  
Author(s):  
Masahiro Tatsuta ◽  
Hiroyuki Mizumoto ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Rna Synthesis ◽  
Red Clover ◽  
Loop Structure ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Protein Coding ◽  
Subgenomic Rna ◽  
Stem Loop Structure ◽  
Rna Interaction

ABSTRACT The expression of the coat protein gene requires RNA-mediated trans-activation of subgenomic RNA synthesis in Red clover necrotic mosaic virus (RCNMV), the genome of which consists of two positive-strand RNAs, RNA1 and RNA2. The trans-acting RNA element required for subgenomic RNA synthesis from RNA1 has been mapped previously to the protein-coding region of RNA2, whereas RNA2 is not required for the replication of RNA1. In this study, we investigated the roles of the protein-coding region in RNA2 replication by analyzing the replication competence of RNA2 mutants containing deletions or nucleotide substitutions. Our results indicate that the same stem-loop structure (SL2) that functions as a trans-activator for RNA-mediated coat protein expression is critically required for the replication of RNA2 itself. Interestingly, however, disruption of the RNA-RNA interaction by nucleotide substitutions in the region of RNA1 corresponding to the SL2 loop of RNA2 does not affect RNA2 replication, indicating that the RNA-RNA interaction is not required for RNA2 replication. Further mutational analysis showed that, in addition to the stem-loop structure itself, nucleotide sequences in the stem and in the loop of SL2 are important for the replication of RNA2. These findings suggest that the structure and nucleotide sequence of SL2 in RNA2 play multiple roles in the virus life cycle.

scholarly journals An approach for the identification of microRNA with an application to Anopheles gambiae.

2006 ◽  
Vol 53 (2) ◽  
pp. 303-309 ◽  
Author(s):  
Raghunath Chatterjee ◽  
Keya Chaudhuri
Keyword(s):  
Drosophila Melanogaster ◽  
Anopheles Gambiae ◽  
Rna Folding ◽  
Noncoding Rnas ◽  
Phylogenetic Distance ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Related Organism ◽  
Post Transcriptional Regulation

MicroRNAs (miRNAs) are an abundant class of 20-27 nt long noncoding RNAs, involved in post-transcriptional regulation of genes in eukaryotes. These miRNAs are usually highly conserved between the genomes of related organisms and their pre-miRNA transcript, about 60-120 nt long, forms extended stem-loop structure. Keeping these facts in mind miRsearch is developed which relies on searching the homologues of all known miRNAs of one organism in the genome of a related organism allowing few mismatches depending on the phylogenetic distance between them, followed by assessing for the capability of formation of stem-loop structure. The precursor sequences so obtained were then screened through the RNA folding program MFOLD selecting the cut-off values on the basis of known Drosophila melanogaster pre-miRNAs. With this approach, about 91 probable candidate miRNAs along with pre-miRNAs were identified in Anopheles gambiae using known D. melanogaster miRNAs. Out of these, 41 probable miRNAs have 100% similarity with already known D. melanogaster miRNAs and others were found to be at least 85% similar to the miRNAs of various other organisms.

968: Gelsolin Gene Silencing Involving Unusual Chromatin Structures and a Novel Stem Loop Structure of the Promoter Region in Human Bladder Cancer

2004 ◽  
Vol 171 (4S) ◽  
pp. 256-257
Author(s):  
Kazunori Haga ◽  
Ataru Sazawa ◽  
Toru Harabayashi ◽  
Nobuo Shinohara ◽  
Minoru Nomoto ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Gene Silencing ◽  
Promoter Region ◽  
Loop Structure ◽  
Human Bladder Cancer ◽  
Human Bladder ◽  
Stem Loop ◽  
Stem Loop Structure

Detection and sequence analysis of an imperfect stem-loop structure in cis activating gene element from SV40PolyA

2011 ◽  
Vol 33 (4) ◽  
pp. 337-346
Author(s):  
Hong-Gang WANG ◽  
Huan MA ◽  
Zhu LI ◽  
Bin ZHANG ◽  
Xiang-Yang JING ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
In Cis ◽  
Gene Element

scholarly journals Identification and in Silico Characterization of Novel and Conserved MicroRNAs in Methyl Jasmonate-Stimulated Scots Pine (Pinus sylvestris L.) Needles

Forests ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 384
Author(s):  
Baiba Krivmane ◽  
Ilze Šņepste ◽  
Vilnis Šķipars ◽  
Igor Yakovlev ◽  
Carl Gunnar Fossdal ◽  
...  
Keyword(s):  
Methyl Jasmonate ◽  
Scots Pine ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Stem Loop ◽  
Protein Coding ◽  
Stem Loop Structure ◽  
In Silico Characterization ◽  
Potential Precursor

MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim of this study was to identify and characterize novel and conserved microRNAs expressed in methyl jasmonate-treated Scots pine needles. In addition, potential precursor sequences and target genes of the identified miRNAs were determined by alignment to the Pinus unigene set. Potential precursor sequences were identified using the miRAtool, conserved miRNA precursors were also tested for the ability to form the required stem-loop structure, and the minimal folding free energy indexes were calculated. By comparison with miRBase, 4975 annotated sequences were identified and assigned to 173 miRNA groups, belonging to a total of 60 conserved miRNA families. A total of 1029 potential novel miRNAs, grouped into 34 families were found, and 46 predicted precursor sequences were identified. A total of 136 potential target genes targeted by 28 families were identified. The majority of previously reported highly conserved plant miRNAs were identified in this study, as well as some conserved miRNAs previously reported to be monocot specific. No conserved dicot-specific miRNAs were identified. A number of potential gymnosperm or conifer specific miRNAs were found, shared among a range of conifer species.

scholarly journals Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting

2021 ◽  
Vol 14 (2) ◽  
pp. 121
Author(s):  
André Miranda ◽  
Tiago Santos ◽  
Eric Largy ◽  
Carla Cruz
Keyword(s):  
Loop Structure ◽  
Binding Properties ◽  
Stem Loop ◽  
Affinity Ligand ◽  
Stem Loop Structure ◽  
Topology Maintenance ◽  
G Quadruplex ◽  
Dimeric Form ◽  
Biophysical Techniques ◽  
Melting Experiments

We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 5′-end that are complementary to nucleotides at the 3′-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.

scholarly journals MicroRNomics: a newly emerging approach for disease biology

2008 ◽  
Vol 33 (2) ◽  
pp. 139-147 ◽  
Author(s):  
Chunxiang Zhang
Keyword(s):  
Gene Expression ◽  
Genomic Medicine ◽  
Noncoding Rnas ◽  
Cellular Tissue ◽  
Tissue Type ◽  
Regulation Of Expression ◽  
Protein Coding ◽  
Small Noncoding Rnas ◽  
Disease Biology ◽  
Genomic Scale

Genomic evidence reveals that gene expression in humans is precisely controlled in cellular, tissue-type, temporal, and condition-specific manners. Completely understanding the regulatory mechanisms of gene expression is therefore one of the most important issues in genomic medicine. Surprisingly, recent analyses of the human and animal genomes have demonstrated that the majority of RNA transcripts are relatively small, noncoding RNAs (sncRNAs), rather than large, protein coding message RNAs (mRNAs). Moreover, these sncRNAs may represent a novel important layer of regulation for gene expression. The most important breakthrough in this new area is the discovery of microRNAs (miRNAs). miRNAs comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. As a group, miRNAs may directly regulate ∼30% of the genes in the human genome. In keeping with the nomenclature of RNomics, which is to study sncRNAs on the genomic scale, “microRNomics” is coined here to describe a novel subdiscipline of genomics that studies the identification, expression, biogenesis, structure, regulation of expression, targets, and biological functions of miRNAs on the genomic scale. A growing body of exciting evidence suggests that miRNAs are important regulators of cell differentiation, proliferation/growth, mobility, and apoptosis. These miRNAs therefore play important roles in development and physiology. Consequently, dysregulation of miRNA function may lead to human diseases such as cancer, cardiovascular disease, liver disease, immune dysfunction, and metabolic disorders. microRNomics may be a newly emerging approach for human disease biology.

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