scholarly journals The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs

2018 ◽  
Author(s):  
Lilli Olgeiser ◽  
Carl Haag ◽  
Susan Boerner ◽  
Jernej Ule ◽  
Anke Busch ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Hyphal Growth ◽  
Binding Motif ◽  
Uv Crosslinking ◽  
Rrm Domain ◽  
Messenger Ribonucleoprotein ◽  
Close Proximity ◽  
Nucleotide Resolution

AbstractRNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3’ untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.

scholarly journals An improved iCLIP protocol

2021 ◽  
Author(s):  
Flora C. Y. Lee ◽  
Anob M. Chakrabarti ◽  
Heike Hänel ◽  
Elisa Monzón-Casanova ◽  
Martina Hallegger ◽  
...  
Keyword(s):  
Cdna Library ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Library Preparation ◽  
Powerful Technique ◽  
Uv Crosslinking ◽  
Nucleotide Resolution ◽  
Cdna Library Preparation

AbstractCrosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

scholarly journals The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy

2021 ◽  
Author(s):  
Javier Larrasa-Alonso ◽  
María Villalba ◽  
Carlos Martí-Gómez ◽  
Paula Ortiz-Sánchez ◽  
Marina López-Olañeta ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Cardiac Hypertrophy ◽  
Diastolic Dysfunction ◽  
Ventricular Hypertrophy ◽  
Transcriptional Activity ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Left Ventricular ◽  
Therapeutic Tools ◽  
Nucleotide Resolution

Rationale: RNA-binding proteins (RBPs) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP serine/arginine-rich splicing factor 4 (SRSF4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. Objective: To investigate the role of SRSF4 in the heart. Methods and Results: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long non-coding RNAs (lncRNAs), including GAS5 (growth arrest specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide-resolution cross-linking and immuno-precipitation (iCLIP). GAS5 is a repressor of the glucocorticoid receptor (GR) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. Conclusions: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in Cushing's syndrome patients.

Molecular dissection of a genome defense pathway in Neurospora crassa

2015 ◽  
Author(s):  
◽  
Erin C. Boone
Keyword(s):  
Neurospora Crassa ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Periphery ◽  
Bimolecular Fluorescence Complementation ◽  
Proper Function ◽  
Rnai Pathway ◽  
Binding Motif ◽  
Perinuclear Region ◽  
A Genome

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Meiotic silencing by unpaired DNA (MSUD) is an RNA interference (RNAi) pathway in Neurospora crassa that detects genes without a homologous partner and silences them for the duration of sexual development. In this study, we have further elucidated the function of known MSUD proteins, identified novel proteins that are required for MSUD, and demonstrated the conservation of RNAi-related processes at the nuclear periphery. We began by showing SAD-2 is crucial for the localization of other MSUD proteins in the perinuclear region. These data suggest that SAD-2 works as a scaffold protein and that proper function of MSUD, like other germline RNAi-like systems, is reliant on the presence of silencing proteins in the perinuclear region. An MSUD suppression assay identified two novel MSUD proteins, SAD-Y and SAD-B'. Even though SAD-Y and its homologs contain a conserved putative RNA- binding motif, they have yet to be assigned to a biochemical pathway. Our work here has linked silencing to SAD-Y-like proteins. SAD-Y has been shown to interact with other MSUD factors in both the nucleus and at the nuclear periphery. SAD-B's homolog has been found in the nuage, an epicenter for RNA-binding proteins involved in post-transcriptional regulation for Drosophila germline cells. SAD-B interacts with core MSUD proteins and has an especially intimate association with SMS-2, which requires it for localization. Furthermore, bimolecular fluorescence complementation (BiFC) revealed that SAD-B' interacts with a Golgi retrograde transport protein and an autophagy marker protein, suggesting the importance of the endomembrane system in this RNAi process.

scholarly journals Posttranscriptional regulation of human endogenous retroviruses by RNA-binding motif protein 4, RBM4

2020 ◽  
Vol 117 (42) ◽  
pp. 26520-26530
Author(s):  
Amir K. Foroushani ◽  
Bryan Chim ◽  
Madeline Wong ◽  
Andre Rastegar ◽  
Patrick T. Smith ◽  
...  
Keyword(s):  
Human Genome ◽  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ectopic Expression ◽  
Transcript Level ◽  
Endogenous Retroviruses ◽  
Host Protein ◽  
Binding Motif ◽  
Sequencing Data

The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts. Most studies of RBPs have concentrated on their action on host protein-encoding mRNAs, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements, termed endogenous retroviruses (ERVs), that comprise ∼8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions, suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown how broadly RBPs and ERV transcripts directly interact to provide a posttranscriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA-sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-binding motif protein 4 (RBM4). We used photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in an elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the LTRs of HERV-K-10, -K-11, and -K-20, and validated the functionality of this site using reporter assays. In summary, we systematically identified RBPs that may regulate ERV function and demonstrate a role for RBM4 in controlling ERV expression.

scholarly journals Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2

2020 ◽  
Vol 48 (7) ◽  
pp. 3869-3887 ◽  
Author(s):  
Linlin Hou ◽  
Yuanjie Wei ◽  
Yingying Lin ◽  
Xiwei Wang ◽  
Yiwei Lai ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Mobility ◽  
Binding Motif ◽  
Dna And Rna ◽  
Somatic Cell Reprogramming ◽  
Double Stranded Dna

Abstract Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA–DNA chimaeras

2013 ◽  
Vol 449 (3) ◽  
pp. 719-728 ◽  
Author(s):  
Lydia Prongidi-Fix ◽  
Laure Schaeffer ◽  
Angelita Simonetti ◽  
Sharief Barends ◽  
Jean-François Ménétret ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Full Length ◽  
Molar Ratio ◽  
Detailed Knowledge ◽  
Histone H4 ◽  
Purification Method

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA–DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

scholarly journals A nuclear localization domain in the hnRNP A1 protein.

1995 ◽  
Vol 129 (3) ◽  
pp. 551-560 ◽  
Author(s):  
H Siomi ◽  
G Dreyfuss
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Hnrnp A1 ◽  
Binding Domains ◽  
Rich Domain ◽  
Localization Domain ◽  
Hnrnp Proteins

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.

Over-expression of a conserved RNA-binding motif (RRM) domain (csRRM2) improves components ofBrassica napusyield by regulating cell size

2012 ◽  
Vol 131 (5) ◽  
pp. 614-619 ◽  
Author(s):  
Weiwei Qi ◽  
Fengqi Zhang ◽  
Fan Sun ◽  
Yongjuan Huang ◽  
Rongzhan Guan ◽  
...  
Keyword(s):  
Cell Size ◽  
Rna Binding ◽  
Binding Motif ◽  
Over Expression ◽  
Rrm Domain

scholarly journals Predicting localized affinity of RNA binding proteins to transcripts with convolutional neural networks

2021 ◽  
Author(s):  
Alexander Kitaygorodsky ◽  
Emily Jin ◽  
Yufeng Shen
Keyword(s):  
Binding Affinity ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Computational Prediction ◽  
Data Sets ◽  
Coding Region ◽  
Protein Coding ◽  
Specific Affinity

RNA binding proteins (RBPs) are important regulators of transcriptional and post-transcriptional processes. Computational prediction of localized RBP binding affinity with transcripts is important for interpretation of genetic variation, especially variants outside of protein coding region. Here we describe POLARIS (Prediction Of Localized Affinity for RBPs In Sequence), a new deep-learning method for achieving fast, site-specific binding affinity predictions of RNA-binding proteins (RBPs) to the transcribed genome. POLARIS has two modules: 1. a convolutional neural network (CNN) to predict overall RBP binding within a region based on transcript sequence content and expression level; 2. a Gradient-weighted Class Activation Mapping (GradCAM) implementation for efficient signal backpropagation to individual sequence positions. We trained the model using enhanced crosslinking and immunoprecipitation (eCLIP) data from ENCODE. POLARIS has good performance with a median AUC ~ 0.96 for 160 RBPs across three different cell lines, substantially higher than selected popular published methods trained and tested on the same data sets. When tested on data from a different cell line with the same RBPs, the overall performance is maintained, supporting the ability of cell-type specific affinity prediction. Finally, the GradCAM module allows the model to identify the informative sites in a region that drive prediction. The localized prediction facilitates interpretation of the results and provides basis for inference of functional impact of noncoding variants.

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