scholarly journals Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

2021 ◽  
Author(s):  
Sarah E Cabral ◽  
Kimberly Mowry
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Binding Domains ◽  
Multivalent Interactions

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

scholarly journals Combinatorial recognition of clustered RNA elements by a multidomain RNA-binding protein, IMP3

2018 ◽  
Author(s):  
Tim Schneider ◽  
Lee-Hsueh Hung ◽  
Masood Aziz ◽  
Anna Wilmen ◽  
Stephanie Thaum ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Binding Domains ◽  
Systematic Strategy ◽  
Rna Elements ◽  
Target Rna

AbstractHow multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

scholarly journals The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

2021 ◽  
Vol 17 (9) ◽  
pp. e1009931
Author(s):  
Jorge Vera-Otarola ◽  
Estefania Castillo-Vargas ◽  
Jenniffer Angulo ◽  
Francisco M. Barriga ◽  
Eduard Batlle ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Dependent Manner ◽  
The Andes ◽  
Viral Nucleocapsid

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

scholarly journals Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

2006 ◽  
Vol 17 (8) ◽  
pp. 3521-3533 ◽  
Author(s):  
Linda D. Kosturko ◽  
Michael J. Maggipinto ◽  
George Korza ◽  
Joo Won Lee ◽  
John H. Carson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Dependent Manner ◽  
Response Element ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Hnrnp E1

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

RNA-BINDING PROTEIN HUR BLOCKADE HAS POTENT ANTI-TUMOR ACTIVITIES IN HUMAN RENAL CELL CARCINOMA BOTH IN VITRO AND IN VIVO

2009 ◽  
Vol 181 (4S) ◽  
pp. 153-153 ◽  
Author(s):  
Sabrina Danilin ◽  
Lionel Thomas ◽  
Thomas Charles ◽  
Carole Sourbier ◽  
Véronique Lindner ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Human Renal Cell Carcinoma

scholarly journals The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

1997 ◽  
Vol 17 (6) ◽  
pp. 3194-3201 ◽  
Author(s):  
R J Buckanovich ◽  
R B Darnell
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
High Affinity ◽  
Loop Region ◽  
Nuclear Rna ◽  
Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

scholarly journals The Polyadenosine RNA Binding Protein ZC3H14 is Required in Mice for Proper Dendritic Spine Density

2020 ◽  
Author(s):  
Stephanie K. Jones ◽  
Jennifer Rha ◽  
Sarah Kim ◽  
Kevin J. Morris ◽  
Omotola F. Omotade ◽  
...  
Keyword(s):  
Dendritic Spine ◽  
Hippocampal Neurons ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Spine Density ◽  
Dendritic Spine Density

AbstractZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), an evolutionarily conserved member of a class of tandem zinc finger (CCCH) polyadenosine (polyA) RNA binding proteins, is associated with a form of heritable, nonsyndromic autosomal recessive intellectual disability. Previous studies of a loss of function mouse model, Zc3h14Δex13/Δex13, provide evidence that ZC3H14 is essential for proper brain function, specifically for working memory. To expand on these findings, we analyzed the dendrites and dendritic spines of hippocampal neurons from Zc3h14Δex13/Δex13 mice, both in situ and in vitro. These studies reveal that loss of ZC3H14 is associated with a decrease in total spine density in hippocampal neurons in vitro as well as in the dentate gyrus of 5-month old mice analyzed in situ. This reduction in spine density in vitro results from a decrease in the number of mushroom-shaped spines, which is rescued by exogenous expression of ZC3H14. We next performed biochemical analyses of synaptosomes prepared from whole wild-type and Zc3h14Δex13/Δex13 mouse brains to determine if there are changes in steady state levels of postsynaptic proteins upon loss of ZC3H14. We found that ZC3H14 is present within synaptosomes and that a crucial postsynaptic protein, CaMKIIα, is significantly increased in these synaptosomal fractions upon loss of ZC3H14. Together, these results demonstrate that ZC3H14 is necessary for proper dendritic spine density in cultured hippocampal neurons and in some regions of the mouse brain. These findings provide insight into how a ubiquitously expressed RNA binding protein leads to neuronal-specific defects that result in brain dysfunction.

scholarly journals YBX1 is required for maintaining myeloid leukemia cell survival by regulating BCL2 stability in an m6A-dependent manner

Blood ◽  
2021 ◽  
Author(s):  
Mengdie Feng ◽  
Xueqin Xie ◽  
Guoqiang Han ◽  
Tiantian Zhang ◽  
Yashu Li ◽  
...  
Keyword(s):  
Cell Survival ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Leukemia Cell ◽  
Dependent Manner ◽  
Myeloid Leukemia Cell

RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly appreciated as being important for normal hematopoiesis and for hematological malignancies as oncogenes or tumor suppressors, essential RBPs for leukemia maintenance and survival remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an m6A-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis, promotes differentiation, coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia (AML) cells in vitro and in vivo. Loss of YBX1 does not obviously affect normal hematopoiesis. Mechanistically, YBX1 interacts with IGF2BPs and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes, and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival due to YBX1 deletion. Thus, our findings uncover a selective and critical role of YBX1 in maintaining myeloid leukemia survival that might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia.

RNA-binding protein CELF1 enhances cell migration, invasion, and chemoresistance by targeting ETS2 in colorectal cancer

2020 ◽  
Vol 134 (14) ◽  
pp. 1973-1990
Author(s):  
Huaiming Wang ◽  
Rongkang Huang ◽  
Wentai Guo ◽  
Xiusen Qin ◽  
Zifeng Yang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Malignant Tumors ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tumor Growth Model

Abstract Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3′-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC.

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