scholarly journals Tho1, a Novel hnRNP, and Sub2 Provide Alternative Pathways for mRNP Biogenesis in Yeast THO Mutants

2006 ◽  
Vol 26 (12) ◽  
pp. 4387-4398 ◽  
Author(s):  
Sonia Jimeno ◽  
Rosa Luna ◽  
María García-Rubio ◽  
Andrés Aguilera
Keyword(s):  
Nuclear Protein ◽  
Rna Binding ◽  
Binding Activity ◽  
Dependent Manner ◽  
Mrna Accumulation ◽  
Alternative Pathways ◽  
Multicopy Suppressors ◽  
Rna Export ◽  
Nascent Mrna ◽  
Mrnp Biogenesis

ABSTRACT THO is a protein complex that functions in cotranscriptional mRNP formation. Yeast THO1 and SUB2 (Saccharomyces cerevisiae) were identified as multicopy suppressors of the expression defects of the hpr1Δ mutant of THO. Here we show that multicopy THO1 suppresses the mRNA accumulation and export defects and the hyperrecombination phenotype of THO mutants but not those of sub2Δ, thp1Δ, or spt4Δ. Similarly, Sub2 overexpression suppresses the RNA export defect of hpr1Δ. Tho1 is a conserved RNA binding nuclear protein that specifically binds to transcribed chromatin in a THO- and RNA-dependent manner and genetically interacts with the shuttling hnRNP Nab2. The ability of Tho1 to suppress hpr1Δ resides in its C-terminal half, which contains the RNA binding activity and is located after a SAP/SAF (scaffold-associated protein/scaffold-associated factor) domain. Altogether, these results suggest that Tho1 is an hnRNP that, similarly to Sub2, assembles onto the nascent mRNA during transcription and participates in mRNP biogenesis and export. Overexpression of Tho1 or Sub2 may provide alternative ways for mRNP formation and export in the absence of a functional THO complex.

scholarly journals Nucleocytoplasmic Shuttling of Polypyrimidine Tract-binding Protein Is Uncoupled from RNA Export

2001 ◽  
Vol 12 (12) ◽  
pp. 3808-3820 ◽  
Author(s):  
Rajesh V. Kamath ◽  
Daniel J. Leary ◽  
Sui Huang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Polypyrimidine Tract ◽  
Nucleocytoplasmic Shuttling ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Rna Export

Polypyrimidine tract binding protein, PTB/hnRNP I, is involved in pre-mRNA processing in the nucleus and RNA localization and translation in the cytoplasm. In this report, we demonstrate that PTB shuttles between the nucleus and cytoplasm in an energy-dependent manner. Deletion mutagenesis demonstrated that a minimum of the N terminus and RNA recognition motifs (RRMs) 1 and 2 are necessary for nucleocytoplasmic shuttling. Deletion of RRM3 and 4, domains that are primarily responsible for RNA binding, accelerated the nucleocytoplasmic shuttling of PTB. Inhibition of transcription directed by either RNA polymerase II alone or all RNA polymerases yielded similar results. In contrast, selective inhibition of RNA polymerase I did not influence the shuttling kinetics of PTB. Furthermore, the intranuclear mobility of GFP-PTB, as measured by fluorescence recovery after photobleaching analyses, increased significantly in transcriptionally inactive cells compared with transcriptionally active cells. These observations demonstrate that nuclear RNA transcription and export are not necessary for the shuttling of PTB. In addition, binding to nascent RNAs transcribed by RNA polymerase II and/or III retards both the nuclear export and nucleoplasmic movement of PTB. The uncoupling of PTB shuttling and RNA export suggests that the nucleocytoplasmic shuttling of PTB may also play a regulatory role for its functions in the nucleus and cytoplasm.

scholarly journals The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury

2021 ◽  
Vol 17 (7) ◽  
pp. e1009494
Author(s):  
Yingying Guo ◽  
Meleana M. Hinchman ◽  
Mercedes Lewandrowski ◽  
Shaun T. Cross ◽  
Danica M. Sutherland ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Entry ◽  
Post Infection ◽  
Rna Binding ◽  
Binding Capacity ◽  
Binding Activity ◽  
Dependent Manner ◽  
Viral Protein Synthesis ◽  
Granule Formation ◽  
Dsrna Binding

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

scholarly journals Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi

2017 ◽  
Vol 114 (52) ◽  
pp. E11208-E11217 ◽  
Author(s):  
Takuya Kajitani ◽  
Hiroaki Kato ◽  
Yuji Chikashige ◽  
Chihiro Tsutsumi ◽  
Yasushi Hiraoka ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Molecular Basis ◽  
Rna Binding ◽  
Noncoding Rnas ◽  
Transcriptional Silencing ◽  
Binding Activity ◽  
Dependent Manner ◽  
Heterochromatin Formation ◽  
A Subunit

Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.

scholarly journals Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins

2002 ◽  
Vol 156 (1) ◽  
pp. 53-64 ◽  
Author(s):  
Amy M. Brownawell ◽  
Ian G. Macara
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Dependent Manner ◽  
Intact Cells ◽  
Double Stranded Rna ◽  
Permeabilized Cells ◽  
Dsrna Binding ◽  
Rna Export

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

scholarly journals ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56

2007 ◽  
Vol 28 (2) ◽  
pp. 601-608 ◽  
Author(s):  
Ichiro Taniguchi ◽  
Mutsuhito Ohno
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Adaptor Proteins ◽  
Rna Helicase ◽  
Specific Protein ◽  
Binding Activity ◽  
Exon Junction Complex ◽  
Dependent Manner ◽  
Insight Into

ABSTRACT Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

scholarly journals The Yeast hnRNP-Like Proteins Yra1p and Yra2p Participate in mRNA Export through Interaction with Mex67p

2001 ◽  
Vol 21 (13) ◽  
pp. 4219-4232 ◽  
Author(s):  
Daniel Zenklusen ◽  
Patrizia Vinciguerra ◽  
Yvan Strahm ◽  
Françoise Stutz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Protein ◽  
Rna Binding ◽  
Mrna Export ◽  
Deletion Mutants ◽  
Variable Regions ◽  
Evolutionarily Conserved ◽  
Rna Export

ABSTRACT Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessentialSaccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)+ RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

scholarly journals The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation to allow viral replication and myocardial injury

2021 ◽  
Author(s):  
Yingying Guo ◽  
Meleana M Hinchman ◽  
Mercedes Lewandrowski ◽  
Shaun T Cross ◽  
Danica M Sutherland ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Entry ◽  
Post Infection ◽  
Rna Binding ◽  
Binding Capacity ◽  
Binding Activity ◽  
Dependent Manner ◽  
Viral Protein Synthesis ◽  
Granule Formation ◽  
Dsrna Binding

The mammalian orthoreovirus double-stranded (ds) RNA binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection remains largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of 13 σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are produced at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not form SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

scholarly journals E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs

1998 ◽  
Vol 72 (10) ◽  
pp. 7960-7971 ◽  
Author(s):  
Stefan Gabler ◽  
Holger Schütt ◽  
Peter Groitl ◽  
Hans Wolf ◽  
Thomas Shenk ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein A ◽  
Adenovirus Type ◽  
Nucleocytoplasmic Transport ◽  
Cellular Protein ◽  
Binding Activity ◽  
Rna Transport ◽  
Mrna Accumulation ◽  
Genes Encoding ◽  
Infected Cells

ABSTRACT The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.

scholarly journals C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

2021 ◽  
Author(s):  
Eun Seon Kim ◽  
Chang Geon Chung ◽  
Jeong Hyang Park ◽  
Byung Su Ko ◽  
Sung Soon Park ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Heterozygous Mutation ◽  
Pathological Feature ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Drosophila Model ◽  
Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

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