scholarly journals Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vandita D Bhat ◽  
Kathleen L McCann ◽  
Yeming Wang ◽  
Dallas R Fonseca ◽  
Tarjani Shukla ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Rna Binding ◽  
Biological Function ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Regulatory Protein ◽  
Motif Length ◽  
C Elegans ◽  
Preferential Binding

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

scholarly journals Identification of proteins and miRNAs that specifically bind an mRNA in vivo

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kathrin Theil ◽  
Koshi Imami ◽  
Nikolaus Rajewsky
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Noncoding Rnas ◽  
Shotgun Proteomics ◽  
Small Rna Sequencing ◽  
C Elegans ◽  
Specific Transcript ◽  
Proteomics Approach

Abstract Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

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.

scholarly journals PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans

2011 ◽  
Vol 192 (6) ◽  
pp. 929-937 ◽  
Author(s):  
Momoyo Hanazawa ◽  
Masafumi Yonetani ◽  
Asako Sugimoto
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Function Analysis ◽  
General Mechanism ◽  
Granule Formation ◽  
C Elegans ◽  
Germ Granules ◽  
Self Association

Germ granules are germ lineage–specific ribonucleoprotein (RNP) complexes, but how they are assembled and specifically segregated to germ lineage cells remains unclear. Here, we show that the PGL proteins PGL-1 and PGL-3 serve as the scaffold for germ granule formation in Caenorhabditis elegans. Using cultured mammalian cells, we found that PGL proteins have the ability to self-associate and recruit RNPs. Depletion of PGL proteins from early C. elegans embryos caused dispersal of other germ granule components in the cytoplasm, suggesting that PGL proteins are essential for the architecture of germ granules. Using a structure–function analysis in vivo, we found that two functional domains of PGL proteins contribute to germ granule assembly: an RGG box for recruiting RNA and RNA-binding proteins and a self-association domain for formation of globular granules. We propose that self-association of scaffold proteins that can bind to RNPs is a general mechanism by which large RNP granules are formed.

scholarly journals HNRNPH1 Is a Novel Regulator Of Cellular Proliferation and Disease Progression in Chronic Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Menghan Liu ◽  
Lin Yang ◽  
Xiaojun Liu ◽  
Ziyuan Nie ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Cellular Proliferation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Multiple Cancer

RNA binding proteins act as essential modulators in cancers by regulating biological cellular processes. Heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1), as a key member of the heterogeneous nuclear ribonucleoproteins family, is frequently upregulated in multiple cancer cells and involved in tumorigenesis. However, the function of HNRNPH1 in chronic myeloid leukemia (CML) remains unclear. In the present study, we revealed that HNRNPH1 expression level was upregulated in CML patients and cell lines. Moreover, the higher level of HNRNPH1 was correlated with disease progression of CML. In vivo and in vitro experiments showed that knockdown of HNRNPH1 inhibited cell proliferation and promoted cell apoptosis in CML cells. Importantly, knockdown of HNRNPH1 in CML cells enhanced sensitivity to imatinib. Mechanically, HNRNPH1 could bind to the mRNA of PTPN6 and negatively regulated its expression. PTPN6 mediated the regulation between HNRNPH1 and PI3K/AKT activation. Furthermore, the HNRNPH1–PTPN6–PI3K/AKT axis played a critical role in CML tumorigenesis and development. The present study first investigated the deregulated HNRNPH1–PTPN6–PI3K/AKT axis moderated cell growth and apoptosis in CML cells, whereby targeting this pathway may be a therapeutic CML treatment.

scholarly journals YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

2015 ◽  
Vol 208 (7) ◽  
pp. 913-929 ◽  
Author(s):  
Syam Prakash Somasekharan ◽  
Amal El-Naggar ◽  
Gabriel Leprivier ◽  
Hongwei Cheng ◽  
Shamil Hajee ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Granule Formation ◽  
Ribonucleoprotein Complexes ◽  
Highly Correlated ◽  
As Stress

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

Abstract 221: Sam68 Interacts With mTORC1 and Contributes to Myocardial Hypertrophy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Junlan Zhou ◽  
Chan Boriboun ◽  
Gangjian Qin
Keyword(s):  
Cardiac Hypertrophy ◽  
Wall Thickness ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Left Ventricular ◽  
Heart Weight ◽  
Continuous Administration ◽  
Chamber Size

Introduction: The Src-associated in mitosis 68 kDa (Sam68) belongs to the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. Our recent work revealed its critical role in the regulation of adipose thermogenesis. However, whether Sam68 regulates cardiomyocyte biology and heart disease is currently unknown. Methods and Results: Echocardiography analyses were performed in Sam68 -/- mice and WT littermates; the baseline left-ventricular wall thickness (LVPWs and LVPWd), chamber size (LVDs and LVDd), and contractility (LVEF and FS) are similar between the 2 groups of animals. However, after transverse aortic constriction (TAC) for 2 and 4 weeks, Sam68 -/- mice exhibited a significantly lesser increase in LV wall thickness (at 2 and 4 weeks), a closer to normal LV chamber size (at 4 weeks), and a lower heart-weight (HW)/body-weight (BW) ratio (at 4 weeks), as compared to WT controls. Consistently, after continuous administration of angiotensin II (by subcutaneous osmotic minipumps) for 2 weeks, Sam68 -/- mice displayed a similarly ameliorated LV wall thickness, chamber size, and HW/BW ratio. The attenuated hypertrophic responses of Sam68 -/- mice to both TAC and Ang II treatment was supported by a lowered expression of fetal genes (β-myosin heavy chain, atrial and brain natriuretic peptides) in the heart (qRT-PCR) and excitingly, by the decreased activity of mTORC1, as indicated by the lowered levels of phosphorylated S6K1 and 4E-BP1 (Western blotting). The cardiomyocyte-specific role of Sam68 was further confirmed in cultured H9c2 cells with lentivirus-mediated Sam68 knockdown, which confirmed the reduction of mTORC1 activity. Furthermore, co- immunoprecipitation assays revealed that the endogenous Sam68 protein interacts Raptor and mTOR, but not Rictor, in the mTOR complex. Experiments are underway to establish the functional significance of the Sam68-mTOR pathway in cardiac hypertrophy and failure in vivo. Conclusion: Collectively, our data suggest that Sam68 is a novel cofactor of mTORC1. It interacts with Raptor to augment mTOR signaling and contribute to the development of cardiac hypertrophy.

scholarly journals RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA

2020 ◽  
pp. jbc.RA120.014894
Author(s):  
Ravi Kumar ◽  
Dipak Kumar Poria ◽  
Partho Sarothi Ray
Keyword(s):  
Inflammatory Response ◽  
Chronic Inflammation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Suppressor Gene ◽  
Cooperative Action ◽  
Translation Repression

Post-transcriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a pro-inflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) HuR in response to LPS stimulation, but the role of other regulatory factors remain unknown. Here we report that the RBP Lupus antigen (La) interacts with the 3’UTR of PDCD4 mRNA and prevents miR-21-mediated translation repression. While LPS causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

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