scholarly journals Upf1/Upf2 Regulation of 3′ Untranslated Region Splice Variants of AUF1 Links Nonsense-Mediated and A+U-Rich Element-Mediated mRNA Decay

2006 ◽  
Vol 26 (23) ◽  
pp. 8743-8754 ◽  
Author(s):  
Lili Banihashemi ◽  
Gerald M. Wilson ◽  
Neha Das ◽  
Gary Brewer
Keyword(s):  
Mammalian Cells ◽  
Mrna Decay ◽  
Rna Binding ◽  
Splice Variants ◽  
Dominant Negative ◽  
Enhanced Expression ◽  
Nonsense Mediated Mrna Decay ◽  
Translational Termination ◽  
Development Proceeds ◽  
In Cells

ABSTRACT AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3′-untranslated regions (3′-UTRs). Previous work suggested that this heterogeneity in 3′-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3′-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3′-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3′-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3′-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3′-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3′-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.

scholarly journals Regulation of Multiple Core Spliceosomal Proteins by Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay

2008 ◽  
Vol 28 (13) ◽  
pp. 4320-4330 ◽  
Author(s):  
Arneet L. Saltzman ◽  
Yoon Ki Kim ◽  
Qun Pan ◽  
Matthew M. Fagnani ◽  
Lynne E. Maquat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mrna Decay ◽  
Splice Variants ◽  
Cellular Functions ◽  
Regulate Gene Expression ◽  
Premature Termination Codons ◽  
Microarray Profiling ◽  
Nonsense Mediated Mrna Decay ◽  
Regulate Gene

ABSTRACT Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

2008 ◽  
Vol 414 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Richard M. van Rijn ◽  
André van Marle ◽  
Paul L. Chazot ◽  
Ellen Langemeijer ◽  
Yongjun Qin ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Inflammatory Diseases ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Histamine H4 Receptor ◽  
Negative Effect ◽  
H4 Receptor

The H4R (histamine H4 receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H3R. The occurrence of at least twenty different hH3R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R–ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the surface expression of H4R(390). We detected H4R(390)–H4R splice varianthetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H4R splice variants were detected in various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H4R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization.

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.

Nonsense-mediated mRNA decay — Mechanisms of substrate mRNA recognition and degradation in mammalian cells

2013 ◽  
Vol 1829 (6-7) ◽  
pp. 612-623 ◽  
Author(s):  
Christoph Schweingruber ◽  
Simone C. Rufener ◽  
David Zünd ◽  
Akio Yamashita ◽  
Oliver Mühlemann
Keyword(s):  
Mammalian Cells ◽  
Mrna Decay ◽  
Nonsense Mediated Mrna Decay

scholarly journals Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphoid Cells

2020 ◽  
Author(s):  
Jean-Marie Lambert ◽  
Mohamad Omar Ashi ◽  
Nivine Srour ◽  
Laurent Delpy ◽  
Jérôme Saulière
Keyword(s):  
Quality Control ◽  
Mrna Decay ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Surveillance Systems ◽  
Rna Surveillance ◽  
Accelerated Degradation ◽  
Premature Termination Codons ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Therefore, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we will describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphoid cells.

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.

scholarly journals Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.

1996 ◽  
Vol 16 (10) ◽  
pp. 5491-5506 ◽  
Author(s):  
Y Weng ◽  
K Czaplinski ◽  
S W Peltz
Keyword(s):  
Mrna Decay ◽  
Rna Binding ◽  
Biochemical Characterization ◽  
Translation Termination ◽  
Nonsense Suppression ◽  
Nucleoside Triphosphate ◽  
Rich Region ◽  
Amino Terminal ◽  
Nonsense Mediated Mrna Decay

To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates the degradation of mRNAs. In the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in the stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. In the work described in the accompanying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol. 16:5477-5490, 1996) mutations in the helicase region of Upf1p that inactivated its mRNA decay function but prevented suppression of leu2-2 and tyr7-1 nonsense alleles are identified. On the basis of these results, we suggested that Upf1p is a multifunctional protein involved in modulating mRNA decay and translation termination at nonsense codons. If this is true, we predict that UPF1 mutations with the converse phenotype should be identified. In this report, we describe the identification and biochemical characterization of mutations in the amino-terminal cysteine- and histidine-rich region of Upf1p that have normal nonsense-mediated mRNA decay activities but are able to suppress leu2-2 and tyr7-1 nonsense alleles. Biochemical characterization of these mutant proteins demonstrated that they have altered RNA binding properties. Furthermore, using the two-hybrid system, we characterized the Upf1p-Upf2p interactions and demonstrated that Upf2p interacts with Upf3p. Mutations in the cysteine- and histidine-rich region of Upf1p abolish Upf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.

scholarly journals Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphocytes

2020 ◽  
Vol 21 (4) ◽  
pp. 1335 ◽  
Author(s):  
Jean-Marie Lambert ◽  
Mohamad Omar Ashi ◽  
Nivine Srour ◽  
Laurent Delpy ◽  
Jérôme Saulière
Keyword(s):  
Quality Control ◽  
Alternative Splicing ◽  
Mrna Decay ◽  
Dominant Negative ◽  
Surveillance Systems ◽  
Rna Surveillance ◽  
Accelerated Degradation ◽  
Premature Termination Codons ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid the synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Thus, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphocytes.

scholarly journals Insights into the assembly and architecture of a Staufen-mediated mRNA decay (SMD)-competent mRNP

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Manjeera Gowravaram ◽  
Juliane Schwarz ◽  
Sana K. Khilji ◽  
Henning Urlaub ◽  
Sutapa Chakrabarti
Keyword(s):  
Catalytic Activity ◽  
Cross Talk ◽  
Untranslated Region ◽  
Mrna Decay ◽  
Molecular Mechanisms ◽  
Rna Helicase ◽  
Secondary Structures ◽  
Nonsense Mediated Mrna Decay ◽  
In Cells

Abstract The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3’-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells.

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