The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction

Abstract The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.

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AU-rich RNA binding proteins in hematopoiesis and leukemogenesis

Blood ◽

10.1182/blood-2011-07-347237 ◽

2011 ◽

Vol 118 (22) ◽

pp. 5732-5740 ◽

Cited By ~ 35

Author(s):

Maria Baou ◽

John D. Norton ◽

John J. Murphy

Keyword(s):

Cell Growth ◽

Regulatory Networks ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Growth And Survival ◽

Hematopoietic Malignancies ◽

Gene Regulatory

Abstract Posttranscriptional mechanisms are now widely acknowledged to play a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. Although much attention has focused on microRNAs as regulators of mRNA stability/translation, recent data have highlighted the role of several diverse classes of AU-rich RNA-binding protein in the regulation of mRNA decay/stabilization. AU-rich elements are found in the 3′-untranslated region of many mRNAs that encode regulators of cell growth and survival, such as cytokines and onco/tumor-suppressor proteins. These are targeted by a burgeoning number of different RNA-binding proteins. Three distinct types of AU-rich RNA binding protein (ARE poly-U–binding degradation factor-1/AUF1, Hu antigen/HuR/HuA/ELAVL1, and the tristetraprolin/ZFP36 family of proteins) are essential for normal hematopoiesis. Together with 2 further AU-rich RNA-binding proteins, nucleolin and KHSRP/KSRP, the functions of these proteins are intimately associated with pathways that are dysregulated in various hematopoietic malignancies. Significantly, all of these AU-rich RNA-binding proteins function via an interconnected network that is integrated with microRNA functions. Studies of these diverse types of RNA binding protein are providing novel insight into gene-regulatory mechanisms in hematopoiesis in addition to offering new opportunities for developing mechanism-based targeted therapeutics in leukemia and lymphoma.

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Interleukin-6 (IL-6) Enhances C-MYC Translation IN MULTIPLE MYELOMA (MM) CELLS: ROLE of IL-6-INDUCED EFFECTS On the C-MYC RNA-Binding PROTEIN, HNRNPA1.

Blood ◽

10.1182/blood.v114.22.3839.3839 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3839-3839

Author(s):

Yijiang Shi ◽

Joseph Gera ◽

Alan Lichtenstein

Keyword(s):

Cell Growth ◽

Protein Expression ◽

Rna Binding ◽

Conflicts Of Interest ◽

Binding Protein ◽

Rna Binding Protein ◽

Dominant Negative ◽

Start Codon ◽

Cytoplasmic Localization ◽

Mtor Inhibition

Abstract Abstract 3839 Poster Board III-775 Our previous work (Cancer Research 68:10215, 2008) demonstrated that IL-6 enhanced c-myc protein expression in MM cells and function of the RNA-binding protein, hnRNPA1 (A1), was required. This occurred by way of enhanced cap-independent translation mediated via the internal ribosome entry site (IRES) in the 5'UTR of the myc RNA. IRES-dependent translation is the fail safe mechanism for protein expression when cap-dependent translation is suppressed by mTOR inhibition (with curtailed RNA cap-ribosome binding) and is especially important when an mRNA leader is relatively long and highly structured, such that scanning ribosomes are unlikely to efficiently initiate translation. The IRES directly recruits the ribosome to within close proximity to the start codon, bypassing the need for cap binding and ribosome scanning. HNRNPA1 (A1) is a documented IRES trans-acting factor (ITAF) for the myc IRES, facilitating translation but how its effects are enhanced by IL-6 is unknown. HNRNPA1 is a shuttling protein which binds the myc RNA in the nucleus and transports it to the cytoplasm. Initial experiments demonstrated that IL-6 stimulation of ANBL-6 and OCI-My5 MM cell lines, as well as several primary MM specimens, significantly increased the cytoplasmic localization of A1. To test if this enhanced cytoplasmic localization was critical, we stably expressed a dominant negative (DN) A1 gene that is incapable of nuclear-to-cytoplasmic shuttling and which also prevents endogenous hnRNPA1 shuttling. The DN also prevented A1-mediated transport of the shuttling protein, FUS. Expression of the DN in ANBL-6 cells prevented IL-6-induced effects on myc expression and on ANBL-6 cell growth. We also tested if IL-6 treatment affected A1 binding to the myc RNA by immunoprecipitating A1 and performing real time PCR on the immunoprecipitate for myc RNA levels. A significant increase in A1-myc RNA binding was confirmed. Mass spectroscopy demonstrated that IL-6 induced phosphorylation of A1 in its RNA-binding domain, which possibly mediated the enhanced binding. These results demonstrate that, by enhanced binding of the myc ITAF, hnRNPA1, to the myc IRES, and by enhanced transport of the complex to the translationally active cytoplasmic subcellular site, IL-6 may stimulate c-myc translation and subsequent MM cell growth. Disclosures: No relevant conflicts of interest to declare.

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The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

The Journal of Cell Biology ◽

10.1083/jcb.200701005 ◽

2007 ◽

Vol 176 (7) ◽

pp. 929-939 ◽

Cited By ~ 182

Author(s):

Maria Paola Paronetto ◽

Tilman Achsel ◽

Autumn Massiello ◽

Charles E. Chalfant ◽

Claudio Sette

Keyword(s):

Alternative Splicing ◽

Tyrosine Phosphorylation ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Live Cells ◽

Hnrnp A1 ◽

Splice Site Selection ◽

Subnuclear Localization ◽

Mrna Targets

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.

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Critical role of tristetraprolin and AU‐rich element RNA‐binding protein 1 in the suppression of cancer cell growth by globular adiponectin

FEBS Open Bio ◽

10.1002/2211-5463.12541 ◽

2018 ◽

Vol 8 (12) ◽

pp. 1964-1976 ◽

Cited By ~ 1

Author(s):

Nirmala Tilija Pun ◽

Amrita Khakurel ◽

Aastha Shrestha ◽

Sang‐Hyun Kim ◽

Pil‐Hoon Park

Keyword(s):

Cell Growth ◽

Cancer Cell ◽

Rna Binding ◽

Binding Protein ◽

Critical Role ◽

Rna Binding Protein ◽

Cancer Cell Growth ◽

Globular Adiponectin

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Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD

Journal of Biological Chemistry ◽

10.1074/jbc.m114.599399 ◽

2015 ◽

Vol 290 (26) ◽

pp. 16357-16371 ◽

Cited By ~ 29

Author(s):

Natasha M. Sosanya ◽

Luisa P. Cacheaux ◽

Emily R. Workman ◽

Farr Niere ◽

Nora I. Perrone-Bizzozero ◽

...

Keyword(s):

Protein Kinase ◽

Rna Binding ◽

Binding Protein ◽

Mammalian Target Of Rapamycin ◽

Rna Binding Protein ◽

Target Of Rapamycin ◽

Dendritic Branch ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein

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Identification of cellular mRNA targets for RNA-binding protein Sam68

Nucleic Acids Research ◽

10.1093/nar/gkf673 ◽

2002 ◽

Vol 30 (24) ◽

pp. 5452-5464 ◽

Cited By ~ 45

Author(s):

M. Itoh

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Mrna Targets

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Phosphoregulation provides specificity to biomolecular condensates in the cell cycle and cell polarity

The Journal of Cell Biology ◽

10.1083/jcb.201910021 ◽

2020 ◽

Vol 219 (7) ◽

Cited By ~ 2

Author(s):

Therese M. Gerbich ◽

Grace A. McLaughlin ◽

Katelyn Cassidy ◽

Scott Gerber ◽

David Adalsteinsson ◽

...

Keyword(s):

Cell Cycle ◽

Nucleic Acids ◽

Cell Polarity ◽

Filamentous Fungus ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Ashbya Gossypii ◽

Mrna Targets ◽

Functional Identities

Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.

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