The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Mihaela Zavolan - One of the best experts on this subject based on the ideXlab platform.
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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core Proteins and small RNA sequencing.
Genome biology, 2013Co-Authors: Shivendra Kishore, Andreas Gruber, Dominik J. Jedlinski, Afzal Pasha Syed, Hadi Jorjani, Mihaela ZavolanAbstract:Background In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 Protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated Proteins.
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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core Proteins and small RNA sequencing
Genome Biology, 2013Co-Authors: Shivendra Kishore, Dominik J. Jedlinski, Afzal Pasha Syed, Hadi Jorjani, Andreas R Gruber, Mihaela ZavolanAbstract:Background In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 Protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated Proteins. Results Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 Protein. Conclusions We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
Ben Berkhout - One of the best experts on this subject based on the ideXlab platform.
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Boosting AgoshRNA activity by optimized 5'-terminal nucleotide selection.
RNA biology, 2019Co-Authors: Zongliang Gao, Ben Berkhout, Elena Herrera-carrilloAbstract:RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 Protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5'-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5'-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes.
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the influence of the 5 terminal nucleotide on agoshrna activity and biogenesis importance of the polymerase iii transcription initiation site
Nucleic Acids Research, 2017Co-Authors: Elena Herreracarrillo, Zongliang Gao, Alex Harwig, Matthias T Heemskerk, Ben BerkhoutAbstract:Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 Protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.
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The influence of the 5′-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site
Nucleic acids research, 2016Co-Authors: Elena Herrera-carrillo, Zongliang Gao, Alex Harwig, Matthias T Heemskerk, Ben BerkhoutAbstract:Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 Protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.
Shivendra Kishore - One of the best experts on this subject based on the ideXlab platform.
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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core Proteins and small RNA sequencing.
Genome biology, 2013Co-Authors: Shivendra Kishore, Andreas Gruber, Dominik J. Jedlinski, Afzal Pasha Syed, Hadi Jorjani, Mihaela ZavolanAbstract:Background In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 Protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated Proteins.
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Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core Proteins and small RNA sequencing
Genome Biology, 2013Co-Authors: Shivendra Kishore, Dominik J. Jedlinski, Afzal Pasha Syed, Hadi Jorjani, Andreas R Gruber, Mihaela ZavolanAbstract:Background In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 Protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated Proteins. Results Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 Protein. Conclusions We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
Tobias Restle - One of the best experts on this subject based on the ideXlab platform.
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Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Sarah Willkomm, Tobias RestleAbstract:Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.
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A Single Inter-Domain Salt Bridge within the Human Argonaute 2 Protein Crucially Affects Protein Folding and Consequently Enzymatic Activity
Biophysical Journal, 2014Co-Authors: Munishikha Kalia, Sarah Willkomm, Jens Christian Claussen, Alexandre M. J. J. Bonvin, Tobias RestleAbstract:RNA interference is a highly complex process involved in posttranscriptional gene regulation. Argonaute (Ago) Proteins are the key component of the RNA-induced silencing complex (RISC) and mediate RNA interference (RNAi) in association with small RNAs. Ago2 Proteins are composed of four domains: N, PAZ, Mid and PIWI, tethered by L1 and L2 linker regions [1, 2].In the binary complex of Ago2 and single stranded RNA, the 3'-end of the RNA is bound to the PAZ domain and the 5'-phosphate is anchored within a binding pocket in the Mid domain. The PIWI domain harbors the active site, which is composed of a catalytic tetrad as observed in RNase H.In the present study, we performed MD simulations on the hAgo2 Protein in complex with a bound miRNA (pdb 4F3T). The Protein undergoes prominent breathing motions dominated largely by movements of the PAZ and N domains. Most interestingly, we observed the transient formation a thitherto-undescribed inter-domain salt bridge. The introduction of a mutation preventing formation of this salt bridge drastically affected nucleic acid binding properties of recombinant hAgo2 [3] and abolished the enzymatic activity. In summary, the missing salt bridge not only affects overall Protein conformation and stability but also seems to be crucial for proper positioning of the small RNA.1. Elkayam, E., et al., (2012) The structure of human Argonaute-2 in complex with miR-20a. Cell 150(1), 100-110.2. Schirle, N.T. and MacRae, I.J., (2012) The crystal structure of human Argonaute2. Science 336(6084), 1037-1040.3. Deerberg, A., et al., (2013) Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 Protein. PNAS in press.
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Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 Protein
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Andrea Deerberg, Sarah Willkomm, Tobias RestleAbstract:Argonaute (Ago) Proteins are the key component of the RNA-induced silencing complex and mediate RNA interference (RNAi) in association with small RNAs. Although overall the mechanism of RNAi is well understood, many molecular details of this complex process are not. Here we report about in-depth steady-state and, in particular, pre-steady-state characterization of siRNA binding, target RNA recognition, sequence-specific cleavage and product release by recombinant human Ago 2 (hAgo2). In combining our biochemical studies with crystal structures of bacterial Ago Proteins and of recently released hAgo2, we relate kinetic data to conformational changes along the pathway and propose a comprehensive minimal mechanistic model describing fundamental steps during RNAi. Furthermore, in contrast to the current conception, our hAgo2 preparations are programmable with double-stranded siRNA. Accordingly, the system investigated represents a functional minimal RNA-induced silencing complex.
Zongliang Gao - One of the best experts on this subject based on the ideXlab platform.
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Boosting AgoshRNA activity by optimized 5'-terminal nucleotide selection.
RNA biology, 2019Co-Authors: Zongliang Gao, Ben Berkhout, Elena Herrera-carrilloAbstract:RNA interference (RNAi) can be triggered by synthetic small interfering RNAs (siRNAs) or transgene-expressed short hairpin RNAs (shRNAs). Recent evidence indicates that shRNA molecules, with a relatively short stem and small loop, are processed by Argonaute 2 Protein (Ago2). We named these molecules AgoshRNA as Ago2 is involved in both the processing and the subsequent mRNA-silencing reaction. This alternative processing route yields only a single guide strand, which thus avoids potential off-target effects induced by the passenger strand of a regular shRNA. We recently described that the introduction of a 5'-terminal purine (A or G) and a mismatch at the bottom of the hairpin enhances the AgoshRNA activity. The critical 5'-terminal nucleotide (nt) represents the +1 position of the transcriptional promoter, which influences the transcriptional efficiency and initiation accuracy as demonstrated for the H1 RNA polymerase (Pol) III promoter. These findings highlight the necessity of considering Pol III requirements in the design of optimized AgoshRNA cassettes. In this study, we report the design and expression of potent AgoshRNAs by two other popular Pol III promoters: U6 and 7SK, which were recently reported to have a distinct transcription profile compared to the H1 promoter. We propose general rules for the design and expression of potent AgoshRNA molecules using Pol III cassettes, which should augment the application of novel AgoshRNA reagents for basic research and therapeutic purposes.
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the influence of the 5 terminal nucleotide on agoshrna activity and biogenesis importance of the polymerase iii transcription initiation site
Nucleic Acids Research, 2017Co-Authors: Elena Herreracarrillo, Zongliang Gao, Alex Harwig, Matthias T Heemskerk, Ben BerkhoutAbstract:Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 Protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.
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The influence of the 5′-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site
Nucleic acids research, 2016Co-Authors: Elena Herrera-carrillo, Zongliang Gao, Alex Harwig, Matthias T Heemskerk, Ben BerkhoutAbstract:Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 Protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecules.