scholarly journals Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

scholarly journals cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II

2006 ◽  
Vol 26 (7) ◽  
pp. 2688-2696 ◽  
Author(s):  
Eric J. Steinmetz ◽  
Sarah B. H. Ng ◽  
Joseph P. Cloute ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Nascent Transcript ◽  
Protein Coding ◽  
Pol Ii ◽  
Eukaryotic Genes ◽  
Discrete Surface ◽  
Cleavage And Polyadenylation ◽  
Selection For

ABSTRACT Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3′-end formation in the context of an mRNA 3′ untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.

scholarly journals Identifying the sequence specificities of circRNA-binding proteins based on a capsule network architecture

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhengfeng Wang ◽  
Xiujuan Lei
Keyword(s):  
Binding Sites ◽  
Network Architecture ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Circular Rnas ◽  
Sequence Motifs ◽  
Rna Motifs ◽  
Classification Framework ◽  
Bound Sequence

Abstract Background Circular RNAs (circRNAs) are widely expressed in cells and tissues and are involved in biological processes and human diseases. Recent studies have demonstrated that circRNAs can interact with RNA-binding proteins (RBPs), which is considered an important aspect for investigating the function of circRNAs. Results In this study, we design a slight variant of the capsule network, called circRB, to identify the sequence specificities of circRNAs binding to RBPs. In this model, the sequence features of circRNAs are extracted by convolution operations, and then, two dynamic routing algorithms in a capsule network are employed to discriminate between different binding sites by analysing the convolution features of binding sites. The experimental results show that the circRB method outperforms the existing computational methods. Afterwards, the trained models are applied to detect the sequence motifs on the seven circRNA-RBP bound sequence datasets and matched to known human RNA motifs. Some motifs on circular RNAs overlap with those on linear RNAs. Finally, we also predict binding sites on the reported full-length sequences of circRNAs interacting with RBPs, attempting to assist current studies. We hope that our model will contribute to better understanding the mechanisms of the interactions between RBPs and circRNAs. Conclusion In view of the poor studies about the sequence specificities of circRNA-binding proteins, we designed a classification framework called circRB based on the capsule network. The results show that the circRB method is an effective method, and it achieves higher prediction accuracy than other methods.

scholarly journals Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata

2020 ◽  
Vol 21 (18) ◽  
pp. 6967
Author(s):  
Thomas M. Banks ◽  
Tianfang Wang ◽  
Quinn P. Fitzgibbon ◽  
Gregory G. Smith ◽  
Tomer Ventura
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Spiny Lobsters ◽  
Systemic Rnai ◽  
Gel Mobility Shift

RNA interference (RNAi) has become a widely utilized method for studying gene function, yet despite this many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double-stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi and may be the limiting factor in RNAi capacity for species such as spiny lobsters, where gene silencing is not functional. Incubating sera from a variety of species across phyla with dsRNA led to a gel mobility shift in species in which systemic RNAi has been observed, with this response being absent in species in which systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters and for a better understanding of the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

scholarly journals Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

1995 ◽  
Vol 15 (8) ◽  
pp. 4562-4571 ◽  
Author(s):  
D Immanuel ◽  
H Zinszner ◽  
D Ron
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Drosophila Embryo ◽  
Cdna Clones ◽  
Rna Pol Ii ◽  
Pol Ii

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

scholarly journals Double Stranded RNA Binding Proteins in the Serum Contribute to Systemic RNAi across Phyla – Towards Finding the Missing Link in Achelata

2020 ◽  
Author(s):  
Thomas Banks ◽  
Tianfang Wang ◽  
Quinn Fitzgibbon ◽  
Gregory Smith ◽  
Tomer Ventura
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Spiny Lobsters ◽  
Systemic Rnai ◽  
Gel Mobility Shift

RNA interference (RNAi) has become a widely utilised method to study gene function, yet despite this, many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi, and may be the limiting factor in RNAi capacity for species such as spiny lobsters where gene silencing is not functional. Incubating serum from a variety of species across phyla with dsRNA led to a gel mobility shift in species where systemic RNAi has been observed, with this response being absent in species where systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon, and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters, and better understanding the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

IL-1 beta stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region

1994 ◽  
Vol 267 (3) ◽  
pp. F504-F508 ◽  
Author(s):  
S. K. Srivastava ◽  
T. Tetsuka ◽  
D. Daphna-Iken ◽  
A. R. Morrison
Keyword(s):  
Posttranscriptional Regulation ◽  
Untranslated Region ◽  
Rna Binding ◽  
Mesangial Cells ◽  
Rna Binding Proteins ◽  
Interleukin 1 ◽  
Mobility Shift ◽  
Phosphatase Treatment ◽  
Prostaglandin Endoperoxide Synthase ◽  
Gel Mobility Shift

We stimulated rat mesangial cells for different time intervals with interleukin-1 beta (IL-1 beta) and phorbol 12-myristate 13-acetate, prepared cytoplasmic extracts, and examined these extracts for the presence of RNA binding proteins by gel mobility shift assays. Here we report that the 3'-untranslated region (3'-UNTR) of the prostaglandin endoperoxide synthase II (COX II) gene is responsible for posttranscriptional regulation of the response to IL-1 beta. Two cytosolic transacting factors of 65 and 45 kDa, respectively, have been detected that bind to the 3'-UNTR. Competition with excess RNA and acid phosphatase treatment of the cytoplasmic extract suggest the binding is specific and that phosphorylation is required for these rapid binding events. These experiments suggest that IL-1 beta induces the phosphorylation of cytosolic factors, which bind to the 3'-UNTR of COX II mRNA, and stabilizes the message.

scholarly journals Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence.

1995 ◽  
Vol 15 (4) ◽  
pp. 1953-1960 ◽  
Author(s):  
K Nandabalan ◽  
G S Roeder
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Element ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
Hybrid Genes

The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth. Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells. Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation. The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes. The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency. The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA. Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate. RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron. Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.

scholarly journals Deep neural networks for interpreting RNA binding protein target preferences

2019 ◽  
Author(s):  
Mahsa Ghanbari ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Deep Neural Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Predictive Score ◽  
Sequence Motifs ◽  
Multiple Sources ◽  
Complex Features ◽  
Regulatory Functions

AbstractDeep learning has become a powerful paradigm to analyze the binding sites of regulatory factors including RNA-binding proteins (RBPs), owing to its strength to learn complex features from possibly multiple sources of raw data. However, the interpretability of these models, which is crucial to improve our understanding of RBP binding preferences and functions, has not yet been investigated in significant detail. We have designed a multitask and multimodal deep neural network for characterizing in vivo RBP binding preferences. The model incorporates not only the sequence but also the region type of the binding sites as input, which helps the model to boost the prediction performance. To interpret the model, we quantified the contribution of the input features to the predictive score of each RBP. Learning across multiple RBPs at once, we are able to avoid experimental biases and to identify the RNA sequence motifs and transcript context patterns that are the most important for the predictions of each individual RBP. Our findings are consistent with known motifs and binding behaviors of RBPs and can provide new insights about the regulatory functions of RBPs.

scholarly journals Structural basis of Nrd1–Nab3 heterodimerization

2022 ◽  
Vol 5 (4) ◽  
pp. e202101252
Author(s):  
Belén Chaves-Arquero ◽  
Santiago Martínez-Lumbreras ◽  
Sergio Camero ◽  
Clara M Santiveri ◽  
Yasmina Mirassou ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Basis ◽  
Free Form ◽  
Temperature Sensitive ◽  
Nascent Transcript ◽  
Interaction Domain ◽  
Hydrophobic Residues ◽  
Structural Solutions

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1–Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein–protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1–Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

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