scholarly journals Differential effects of Doxorubicin and Actinomycin D on the stability of RNA binding proteins, RBM10 and RBM5: Actinomycin D promotes the nuclear speckles targeting of RBM10 and RBM5 through the novel structural elements

2019 ◽  
Author(s):  
Koji Nishio ◽  
Shanlou Qiao ◽  
Kung Sang Chang
Keyword(s):  
Tumor Cells ◽  
Actinomycin D ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
A549 Cells ◽  
Binding Motif ◽  
Structural Elements ◽  
Nuclear Speckles ◽  
Highly Sensitive

AbstractBackgroundRNA binding motif (RBM) proteins, RBM10v1, RBM10v2 and RBM5 share a high degree of the conserved domains. So far, the drug-sensitivities of the RBMs in tumor cells have not been fully examined.ObjectiveThe expression profiles of RBM10 and RBM5 in several virus-transformed tumor cells, and the effect of the most established antitumor agents, actinomycin D and doxorubicin, were investigated.Methods and ResultsDoxorubicin and actinomycin D differentially reduced RBM10 and RBM5 protein, respectively in both of HeLa and COS-7 cells. RBM10 protein was highly sensitive to doxorubicin in HeLa, COS-7 and A549 cells. In silico analysis revealed the several sumoylation sites of RBM10 and its sumoylated form could be targeted for the activated ubiquitin proteasome system. Actinomycin D affected the nuclear speckles localization of RBM10 and RBM5 in COS-7 and A549 lung carcinoma cells. Addition of actinomycin D in the culture medium and following culture for 3〜4 hours promoted the prominent nuclear speckles of RBM10v2-GFP and RBM5. Hence, we explored the subnuclear localization of the full length RBM10v2 (852aa) and the amino terminally truncated forms and the responsible structural elements. The amino terminally truncated RBM10v2 [#486-852, #642-852], RBM10v2 [#648-852], RBM10v2 [#681-759, #681-852], and RBM10v2 [#660-852] retained the targeting elements for the nuclear speckles, nucleoplasm, nucleoli and whole nuclei, respectively.ConclusionRBM10 is highly sensitive to doxorubicin. Actinomycin D affects the structural elements of RBM10 and promotes the nuclear speckles targeting. The C-terminal regions: RBM10v2 [#642-647], [#642-659], and [#660-680] play critical roles in the targeting to the subnuclear compartments. SIM and sumoylation sits of RBM10 and PML4 are important for molecular interaction of RBM10 and PML.

scholarly journals Position Specific Alternative Splicing and Gene Expression Profiles Along the Tonotopic Axis of Chick Cochlea

2021 ◽  
Vol 8 ◽  
Author(s):  
Heiyeun Koo ◽  
Jae Yeon Hwang ◽  
Sungbo Jung ◽  
Hyeyoung Park ◽  
Jinwoong Bok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Single Gene ◽  
Gene Expression Profiles ◽  
Binding Motif ◽  
Dependent Manner ◽  
Mrna Isoforms

Alternative splicing (AS) refers to the production of multiple mRNA isoforms from a single gene due to alternative selection of exons or splice sites during pre-mRNA splicing. It is a primary mechanism of gene regulation in higher eukaryotes and significantly expands the functional complexity of eukaryotic organisms, contributing to animal development and disease. Recent studies have shown that AS also influences functional diversity by affecting the transcriptomic and proteomic profiles in a position-dependent manner in a single organ. The peripheral hearing organ, the cochlea, is organized to detect sounds at different frequencies depending on its location along the longitudinal axis. This unique functional configuration, the tonotopy, is known to be facilitated by differential gene expression along the cochlear duct. We profiled transcriptome-wide gene expression and AS changes that occur within the different positions of chick cochlea. These analyses revealed distinct gene expression profiles and AS, including a splicing program that is unique to tonotopy. Changes in the expression of splicing factors PTBP3, ESRP1, and ESRP2 were demonstrated to contribute to position-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS at different positions by different RNA-binding proteins. These data, along with gene ontology (GO) analysis, represent a comprehensive analysis of the dynamic regulation of AS at different positions in chick cochlea.

scholarly journals Genome-wide Identification and Expression Analysis of the YTH Domain-containing RNA-binding Protein Family in Citrus Sinensis

2019 ◽  
Vol 144 (2) ◽  
pp. 79-91 ◽  
Author(s):  
Zhigang Ouyang ◽  
Huihui Duan ◽  
Lanfang Mi ◽  
Wei Hu ◽  
Jianmei Chen ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Citrus Sinensis ◽  
Rna Binding ◽  
Developmental Stages ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Genome Wide ◽  
Yth Domain

In eukaryotic systems, messenger RNA regulations, including splicing, 3′-end formation, editing, localization, and translation, are achieved by different RNA-binding proteins and noncoding RNAs. The YTH domain is a newly identified RNA-binding domain that was identified by comparing its sequence with that of splicing factor YT521-B. Previous study showed that the YTH gene plays an important role in plant resistance to abiotic and biotic stress. In this study, 211 YTH genes were identified in 26 species that represent four major plant lineages. Phylogenetic analysis revealed that these genes could be divided into eight subgroups. All of the YTH genes contain a YT521 domain and have different structures. Ten YTH genes were identified in navel orange (Citrus sinensis). The expression profiles of these CitYTH genes were analyzed in different tissues and at different fruit developmental stages, and CitYTH genes displayed distinct expression patterns under heat, cold, salt, and drought stress. Furthermore, expression of the CitYTH genes in response to exogenous hormones was measured. Nuclear localization was also confirmed for five of the proteins encoded by these genes after transient expression in Nicotiana benthamiana cells. This study provides valuable information on the role of CitYTHs in the signaling pathways involved in environmental stress responses in Citrus.

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 The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1958
Author(s):  
Ella Alkalay ◽  
Chen Gam Ze Letova Refael ◽  
Irit Shoval ◽  
Noa Kinor ◽  
Ronit Sarid ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Viral Infections ◽  
Rna Binding Proteins ◽  
Nuclear Periphery ◽  
Lytic Cycle ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Viral Rnas ◽  
Infected Cells

RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi’s sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles. Since splicing is required for the maturation of certain KSHV transcripts, we suggest that the infected cell does not dismantle nuclear speckles but rearranges their components at the nuclear periphery to possibly serve in splicing and transport of viral RNAs into the cytoplasm.

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.

scholarly journals Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence

2008 ◽  
Vol 389 (3) ◽  
pp. 243-255 ◽  
Author(s):  
Kotb Abdelmohsen ◽  
Yuki Kuwano ◽  
Hyeon Ho Kim ◽  
Myriam Gorospe
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Cellular Senescence ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oxidant Damage ◽  
Regulated Gene Expression

AbstractTo respond adequately to oxidative stress, mammalian cells elicit rapid and tightly controlled changes in gene expression patterns. Besides alterations in the subsets of transcribed genes, two posttranscriptional processes prominently influence the oxidant-triggered gene expression programs: mRNA turnover and translation. Here, we review recent progress in our knowledge of theturnover andtranslationregulatory (TTR) mRNA-bindingproteins (RBPs) that influence gene expression in response to oxidative damage. Specifically, we identify oxidant damage-regulated mRNAs that are targets of TTR-RBPs, we review the oxidant-triggered signaling pathways that govern TTR-RBP function, and we examine emerging evidence that TTR-RBP activity is altered with senescence and aging. Given the potent influence of TTR-RBPs upon oxidant-regulated gene expression profiles, we propose that the senescence-associated changes in TTR-RBPs directly contribute to the impaired responses to oxidant damage that characterize cellular senescence and advancing age.

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.

scholarly journals Secondary structure of pre-mRNA introns for genes in the 15q11-12 locus. Mapping of functionally significant motives for RNA-binding proteins and nucleosome positioning signals

2018 ◽  
Author(s):  
Viya B. Fedoseyeva ◽  
Irina A. Zharinova ◽  
Alexander A. Alexandrov
Keyword(s):  
Secondary Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleosome Positioning ◽  
Nuclear Speckles ◽  
Dominant Type ◽  
Mrna Variants ◽  
Locus Mapping ◽  
Folded Structures

AbstractIn this study, we identified reproducible substructures in the folded structures of long intron RNAs for recursive spliced variants and annotated pre-mRNA for GABRB3 and GABRA5. We mapped the RNA motives recognized by RNA-binding proteins for the specified locus and characterized the area of preferred localization. A comparison of pre-mRNA variants revealed the dominant type of protein potential effects. We determined the structural specifics of RNA in the dense Alu cluster and clarified the analogy of apical substructure to the A-Xist fragment of transcriptional variant. Mapping of the nucleosome potential reveals alternation of strong and weak signals at the 3’-end portion of GABRB3 and clusters of nucleosome positioning signal in the vicinity of the Alu cluster. Distribution of simple oligonucleotides among reproducible substructures revealed an enrichment in Py-tracts; for some of them, this may be considered as a complementary supplement to the Pu-tract enrichment of ncRNA Malat1 as a component of nuclear speckles. The secondary structure elements of bidirectional transcripts are predisposed for somatic homolog pairing in this locus, as was previously shown experimentally.A model of potential intron RNA influence on splicing has been suggested based on its interaction with Py-tract-binding RNP, serine-arginine SRSF proteins, ncRNA Malat1, as well as the action of Alu cluster.

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.

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