The mRNA decapping complex is buffered by nuclear localization

2021 ◽  
Vol 134 (18) ◽  
Author(s):  
Kiril Tishinov ◽  
Anne Spang
Keyword(s):  
Nuclear Localization ◽  
Mrna Decay ◽  
Rna Binding ◽  
Dynamic Equilibrium ◽  
Local Concentration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Localization Signals ◽  
P Bodies ◽  
Translational Repressor ◽  
Readily Releasable Pool

ABSTRACT mRNA decay is a key step in regulating the cellular proteome. Processing bodies (P-bodies) are thought to be sites of mRNA decay and/or storage. P-body units assemble into P-body granules under stress conditions. How this assembly is regulated, however, remains poorly understood. Here, we show, in the yeast Saccharomyces cerevisiae, that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by sequestering the Dcp1–Dcp2 (denoted Dcp1/2) decapping complex in the cytoplasm and preventing it from becoming imported into the nucleus by the karyopherin β protein Kap95. One of two nuclear localization signals in Dcp2 overlaps with the RNA-binding site, suggesting an additional mechanism to regulate Dcp1/2 localization. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a readily releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Edc3 to drive phase separation and hence P-body formation.

scholarly journals Decapping complex is essential for functional P-body formation and is buffered by nuclear localization

2020 ◽  
Author(s):  
Kiril Tishinov ◽  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Nuclear Localization ◽  
Mrna Decay ◽  
Dynamic Equilibrium ◽  
Local Concentration ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Translational Repressor

AbstractmRNA decay is a key step in regulating the cellular proteome. Cytoplasmic mRNA is largely turned over in processing bodies (P-bodies). P-body units assemble to form P-body granules under stress conditions. How this assembly is regulated, however, remains still poorly understood. Here, we show that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by capturing the Dcp1/2 decapping complex and preventing it from becoming imported into the nucleus by the karyopherin ß Kap95. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a ready releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3 and Scd6. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Scd6 and Edc3 to drive phase separation and hence P-body formation.

scholarly journals Nonclassical nuclear localization signals mediate nuclear import of CIRBP

2020 ◽  
Vol 117 (15) ◽  
pp. 8503-8514 ◽  
Author(s):  
Benjamin Bourgeois ◽  
Saskia Hutten ◽  
Benjamin Gottschalk ◽  
Mario Hofweber ◽  
Gesa Richter ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Interaction ◽  
Stress Granule ◽  
Rich Region ◽  
Nuclear Localization Signals ◽  
Cargo Proteins

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

scholarly journals A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

1991 ◽  
Vol 113 (6) ◽  
pp. 1243-1254 ◽  
Author(s):  
U Stochaj ◽  
M Osborne ◽  
T Kurihara ◽  
P Silver
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Protein Localization ◽  
Binding Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Localization Signals

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

scholarly journals A non-canonical role for the EDC4 decapping factor in regulating MARF1-mediated mRNA decay

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
William R Brothers ◽  
Steven Hebert ◽  
Claudia L Kleinman ◽  
Marc R Fabian
Keyword(s):  
Essential Role ◽  
Mrna Decay ◽  
Rna Binding ◽  
Mrna Turnover ◽  
Core Component ◽  
P Bodies ◽  
Target Mrnas ◽  
Binding Domains ◽  
Bona Fide ◽  
Decapping Enzyme

EDC4 is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3’ UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 impairs MARF1 activity by preventing its LOTUS domains from binding target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs.

scholarly journals Poly(A) polymerase contains multiple functional domains.

1994 ◽  
Vol 14 (5) ◽  
pp. 2946-2957 ◽  
Author(s):  
T Raabe ◽  
K G Murthy ◽  
J L Manley
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Directed Mutagenesis ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

scholarly journals A Novel Set of Nuclear Localization Signals Determine Distributions of the αCP RNA-Binding Proteins

2003 ◽  
Vol 23 (23) ◽  
pp. 8405-8415 ◽  
Author(s):  
Alexander N. Chkheidze ◽  
Stephen A. Liebhaber
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Nuclear Speckles ◽  
Nuclear Localization Signals ◽  
Kh Domain ◽  
Amino Acid Segment

ABSTRACT αCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major αCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each αCP isoform. Immunofluorescence studies revealed three distinct distributions: αCP1 and αCP2 are predominantly nuclear with specific enrichment of αCP1 in nuclear speckles, αCP3 and αCP4 are restricted to the cytoplasm, and αCP2-KL, an αCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for αCP isoforms. αCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in αCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in αCP3 or αCP4. Consistent with mapping studies, deletion of NLS I from αCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of αCP2. These data demonstrate an unexpected complexity in the compartmentalization of αCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of αCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.

Poly(A) polymerase contains multiple functional domains

1994 ◽  
Vol 14 (5) ◽  
pp. 2946-2957
Author(s):  
T Raabe ◽  
K G Murthy ◽  
J L Manley
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Nuclear Proteins ◽  
Directed Mutagenesis ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

scholarly journals 5′ to 3′ mRNA Decay Factors Colocalize with Ty1 Gag and Human APOBEC3G and Promote Ty1 Retrotransposition

2010 ◽  
Vol 84 (10) ◽  
pp. 5052-5066 ◽  
Author(s):  
James A. Dutko ◽  
Alison E. Kenny ◽  
Eric R. Gamache ◽  
M. Joan Curcio
Keyword(s):  
Mrna Decay ◽  
Rna Binding ◽  
Rna Packaging ◽  
Processing Bodies ◽  
Nonsense Mediated Decay ◽  
Translational Machinery ◽  
P Bodies ◽  
Cellular Mrnas ◽  
Ty1 Retrotransposition ◽  
Human Apobec3g

ABSTRACTThe genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of theSaccharomyces cerevisiaeTy1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5′ to 3′ mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5′ to 3′ mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5′ to 3′ decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5′ to 3′ exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5′ to 3′ mRNA decay is essential for the packaging of Ty1 RNA in VLPs.

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