Regulating the Shuttling of Eukaryotic RNA Polymerase II

Croce, Luciano Di

doi:10.1128/mcb.06093-11

Cited by 5 publications

(8 citation statements)

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“…In light of the data presented in Figures 2 and 5 and in recent publications on the GPN proteins and the NLS-containing protein Iwr1 (Di Croce 2011), we propose a cooperative role for these proteins in RNAPII biogenesis and nuclear import. However, it is unclear whether the GPN proteins function primarily in assembly or nuclear import.…”

Section: Resultssupporting

confidence: 67%

Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

et al. 2013

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The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport.

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Section: Resultssupporting

confidence: 67%

Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

et al. 2013

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“…Multiple interactions between human Pol II and the small GTPase GPN1 indicated its involvement in Pol II assembly and nuclear import (12). Npa3, the yeast homolog of GPN1, is required for nuclear localization of yeast Pol II and binds it in a GTP-dependent manner (13), which argues that the mechanism involved in the subcellular localization of Pol II requires the catalytic function of GNP proteins and is conserved from yeast to mammals (14). Two other proteins involved in Pol II biogenesis, Iwr1 and Rtp1, were identified in genetic screens for suppressors of the growth defect caused by depletion of NC2, a negative regulator of mRNA transcription (15,16).…”

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confidence: 99%

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

Cieśla

Makała

Płonka

et al. 2015

Molecular and Cellular Biology

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Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1⌬ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1⌬ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.A ll eukaryotic cells have at least three different RNA polymerases (Pol). Pol I synthesizes the large precursor of rRNA, Pol II produces mainly mRNAs, and Pol III generates tRNAs, 5S rRNA and other small noncoding RNAs.Pol III, which is the focus of this work, is a heteromultimeric protein complex that contains 17 distinct subunits in Saccharomyces cerevisiae. The two largest subunits, C160 and C128, form opposite sides of the active-center cleft and harbor the catalytic activity, and they are related to the ␤= and ␤ components of the ␣ 2 ␤␤= core of bacterial RNA polymerase. Homology to the bacterial ␣ subunit, although less strong, was also observed, with the subunit AC40 common for yeast and Pol III and Pol I. A second ␣-like subunit, AC19, forms a heterodimer with AC40, which is a functional equivalent to the ␣ 2 homodimer in prokaryotes (1). The polymerase core, in addition to the ␣ 2 ␤␤=-like structure, contains six small subunits, which are structurally and functionally conserved from yeast to humans. The small core subunits either bind or bridge the two largest catalytic subunits. In addition to the central core, the remaining subunits of Pol III are organized in heterodimeric or trimeric stalks or subdomains. Significantly, the C37 to C53 and C82 to C34 subdomains, which are stably bound to the RNA Pol III core, resemble Pol II general transcription factors TFIIF and TFIIE (2).Little is known about how polymerase complexes are assembled from the subunits, how they reach their functional destination, and how they dissociate after transcription. In bacteria, the assembly of RNA polymerase starts with the formation of the ␣␣ dimer, which then interacts with the ␤ subunit to yield an ␣␣␤...

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“…Additionally, several proteins were identified that function together in large multi-protein complexes, including subunits of RNA polymerase II (RNAPII), transcription factor II D (TFIID) and Mediator complexes. The identification of RNAPII subunits is encouraging, as RNAPII is already suspected to assemble within the cytoplasm prior to nuclear import [ 42 , 43 , 69 ]. Similar to RNAPII, the assembly of TFIID subunits has been proposed to occur within the cytoplasm, and subsequently enter the nucleus through a piggybacking mechanism.…”

Section: Resultsmentioning

confidence: 99%

“…Intriguingly, several proteins represented within the group of cargo without predicted cNLSs are already known to use piggybacking, and these mainly belong to the RNAPII complex, where assembly has been shown to take place within the cytoplasm prior to nuclear import [ 43 , 69 ]. Additionally, several TAF proteins belonging to the TFIID complex were identified in this group.…”

Section: Discussionmentioning

confidence: 99%

“…With the development of databases such as the Human Protein Atlas (HPA) and Human Reference Interactome (HuRI), as well as the abundance of publicly available high-throughput mass spectrometry data, it may be possible to establish a more accurate picture of nuclear transport mechanisms [ 36 , 37 , 38 , 39 ]. While examples of piggybacking into the nucleus using Imp-α have been documented for several distinct nuclear proteins, widespread identification of potential piggybacking proteins, or estimates of the extent to which this nuclear import strategy is used, remain poorly characterized [ 40 , 41 , 42 , 43 , 44 , 45 ]. Using a collection of resources and NLS prediction programs, we aimed to acquire information regarding the prevalence of piggybacking and the use of alternative nuclear import pathways in eukaryotic cells.…”

Section: Introductionmentioning

confidence: 99%

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Piggybacking on Classical Import and Other Non-Classical Mechanisms of Nuclear Import Appear Highly Prevalent within the Human Proteome

Tessier

MacNeil

Mymryk

2020

Biology

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One of the most conserved cellular pathways among eukaryotes is the extensively studied classical protein nuclear import pathway mediated by importin-α. Classical nuclear localization signals (cNLSs) are recognized by importin-α and are highly predictable due to their abundance of basic amino acids. However, various studies in model organisms have repeatedly demonstrated that only a fraction of nuclear proteins contain identifiable cNLSs, including those that directly interact with importin-α. Using data from the Human Protein Atlas and the Human Reference Interactome, and proteomic data from BioID/protein-proximity labeling studies using multiple human importin-α proteins, we determine that nearly 50% of the human nuclear proteome does not have a predictable cNLS. Surprisingly, between 25% and 50% of previously identified human importin-α cargoes do not have predictable cNLS. Analysis of importin-α cargo without a cNLS identified an alternative basic rich motif that does not resemble a cNLS. Furthermore, several previously suspected piggybacking proteins were identified, such as those belonging to the RNA polymerase II and transcription factor II D complexes. Additionally, many components of the mediator complex interact with at least one importin-α, yet do not have a predictable cNLS, suggesting that many of the subunits may enter the nucleus through an importin-α-dependent piggybacking mechanism.

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Regulating the Shuttling of Eukaryotic RNA Polymerase II

Cited by 5 publications

References 10 publications

Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

Piggybacking on Classical Import and Other Non-Classical Mechanisms of Nuclear Import Appear Highly Prevalent within the Human Proteome

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