scholarly journals Role for Upf2p Phosphorylation in Saccharomyces cerevisiae Nonsense-Mediated mRNA Decay

2006 ◽  
Vol 26 (9) ◽  
pp. 3390-3400 ◽  
Author(s):  
Weirong Wang ◽  
Iván J. Cajigas ◽  
Stuart W. Peltz ◽  
Miles F. Wilkinson ◽  
Carlos I. González
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Nonsense Mediated Mrna Decay ◽  
Higher Eukaryotes ◽  
Nonsense Codons ◽  
First Time

ABSTRACT Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.

scholarly journals Nonsense-mediated mRNA decay factors cure most [PSI+] prion variants

2018 ◽  
Vol 115 (6) ◽  
pp. E1184-E1193 ◽  
Author(s):  
Moonil Son ◽  
Reed B. Wickner
Keyword(s):  
Protein Interactions ◽  
Mrna Decay ◽  
Rna Binding ◽  
Translation Termination ◽  
Amyloid Formation ◽  
Multifunctional Protein ◽  
Nonsense Mediated Mrna Decay ◽  
Nonsense Codons ◽  
Almost All

The yeast prion [PSI+] is a self-propagating amyloid of Sup35p with a folded in-register parallel β-sheet architecture. In a genetic screen for antiprion genes, using the yeast knockout collection,UPF1/NAM7andUPF3, encoding nonsense-mediated mRNA decay (NMD) factors, were frequently detected. Almost all [PSI+] variants arising in the absence of Upf proteins were eliminated by restored normal levels of these proteins, and [PSI+] arises more frequently inupfmutants. Upf1p, complexed with Upf2p and Upf3p, is a multifunctional protein with helicase, ATP-binding, and RNA-binding activities promoting efficient translation termination and degradation of mRNAs with premature nonsense codons. We find that the curing ability of Upf proteins is uncorrelated with these previously reported functions but does depend on their interaction with Sup35p and formation of the Upf1p–Upf2p–Upf3p complex (i.e., the Upf complex). Indeed, Sup35p amyloid formation in vitro is inhibited by substoichiometric Upf1p. Inhibition of [PSI+] prion generation and propagation by Upf proteins may be due to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers. Alternatively, the association of the Upf complex with amyloid filaments may block the addition of new monomers. Our results suggest that maintenance of normal protein–protein interactions prevents prion formation and can even reverse the process.

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

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 SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

scholarly journals SNP rs4142441 and MYC co-modulated long non-coding RNA OSER1-AS1 suppresses non-small cell lung cancer by sequestering RNA-Binding Protein ELAVL1

2020 ◽  
Author(s):  
Weijia Xie ◽  
Youhao Wang ◽  
Yao Zhang ◽  
Ying Xiang ◽  
Na Wu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Rna Binding ◽  
Lung Cancer Risk ◽  
Rna Binding Protein ◽  
Small Cell ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Small Cell Lung

Abstract Background: Single nucleotide polymorphisms (SNPs) and long non-coding RNAs (lncRNAs) have been involved in the process of lung cancer. Following clues given by lung cancer risk-associated SNPs, we aimed to find novel functional lncRNAs as candidate targets in non-small cell lung cancer (NSCLC). Methods: Case-control analyses were performed in 626 cases and 736 controls matched up on sex and age. The lncRNA OSER1-AS1 was identified near a lung cancer risk-associated SNP rs4142441. Kaplan–Meier survival analysis was performed to investigate the association between OSER1-AS1 expression and overall survival. The influence of rs4142441 on the expression level of OSER1-AS1 was confirmed using Luciferase assays. Subsequently, the biological function of OSER1-AS1 was assessed in vitro by cell proliferation, migration, and invasion experiments through gain- and loss-of-function approaches, and in vivo by subcutaneous tumor model and tail vein injection lung metastasis model. ChIP and RIP experiments were carried out to investigate the interaction between transcription factors, RNA-binding proteins, and OSER1-AS1.Results: OSER1-AS1 was down-regulated in tumor tissue and its low expression was significantly associated with poor overall survival among non-smokers in NSCLC patients. Gain- and loss-of-function studies revealed that OSER1-AS1 acted as a tumor suppressor by inhibiting lung cancer cell growth, migration and invasion in vitro. Xenograft tumor assays and metastasis mouse model confirmed that OSER1-AS1 suppressed tumor growth and metastasis in vivo. The promoter of OSER1-AS1 was repressed by MYC, and the 3’-end of OSER1-AS1 was competitively targeted by microRNA hsa-miR-17-5p and RNA-binding protein ELAVL1. Conclusion: Our results indicated that OSER1-AS1 exerted tumor-suppressive functions by acting as an ELAVL1 decoy to keep it away from its target mRNAs. Our findings characterized OSER1-AS1 as a new tumor suppressive lncRNA in NSCLC, suggesting that OSER1-AS1 may be suitable as a potential biomarker for prognosis, and a potential target for treatment.

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 Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products

2020 ◽  
Author(s):  
Aparna Kishor ◽  
Sarah E Fritz ◽  
Nazmul Haque ◽  
Zhiyun Ge ◽  
Ilker Tunc ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Alternative Polyadenylation ◽  
Transcript Level ◽  
Untranslated Regions ◽  
Multiple Tumor ◽  
The Core ◽  
Nonsense Mediated Mrna Decay ◽  
Tumor Types

Abstract Alternative polyadenylation (APA) produces transcript 3′ untranslated regions (3′UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3′UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3′UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3′UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

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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