scholarly journals Transportin-mediated Nuclear Import of Heterogeneous Nuclear RNP Proteins

1997 ◽  
Vol 138 (6) ◽  
pp. 1181-1192 ◽  
Author(s):  
Mikiko C. Siomi ◽  
Paul S. Eder ◽  
Naoyuki Kataoka ◽  
Lili Wan ◽  
Qing Liu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Mrna Processing ◽  
Hnrnp A1 ◽  
Hnrnp Proteins ◽  
Bidirectional Transport ◽  
Acid Domain ◽  
Nuclear Ribonucleoprotein

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase–M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1–A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

scholarly journals Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

1996 ◽  
Vol 16 (5) ◽  
pp. 2350-2360 ◽  
Author(s):  
E F Michelotti ◽  
G A Michelotti ◽  
A I Aronsohn ◽  
D Levens
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Transcription In Vitro ◽  
Nuclear Ribonucleoprotein ◽  
Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta

1997 ◽  
Vol 110 (11) ◽  
pp. 1325-1331 ◽  
Author(s):  
R.A. Fridell ◽  
R. Truant ◽  
L. Thorne ◽  
R.E. Benson ◽  
B.R. Cullen
Keyword(s):  
Nuclear Import ◽  
Hnrnp A1 ◽  
Cellular Cofactor ◽  
Basic Class ◽  
Localization Signal ◽  
In Trans ◽  
Cellular Import ◽  
Nuclear Ribonucleoprotein

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

scholarly journals Functional Conservation of the Transportin Nuclear Import Pathway in Divergent Organisms

1998 ◽  
Vol 18 (7) ◽  
pp. 4141-4148 ◽  
Author(s):  
Mikiko C. Siomi ◽  
Micheline Fromont ◽  
Jean-Christophe Rain ◽  
Lili Wan ◽  
Fan Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nuclear Import ◽  
Mammalian Cells ◽  
Hnrnp A1 ◽  
Reading Frame ◽  
Functional Conservation ◽  
Amino Acid Region ◽  
Acid Domain ◽  
Mrna Binding

ABSTRACT Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription.

1982 ◽  
Vol 257 (10) ◽  
pp. 5779-5788 ◽  
Author(s):  
G A Kassavetis ◽  
E T Butler ◽  
D Roulland ◽  
M J Chamberlin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription

scholarly journals The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

2004 ◽  
Vol 24 (7) ◽  
pp. 2932-2943 ◽  
Author(s):  
Hailing Cheng ◽  
Xiaoyuan He ◽  
Claire Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Temperature Sensitive ◽  
Repeat Protein ◽  
Tightly Coupled ◽  
Cleavage And Polyadenylation ◽  
Wd Repeat

ABSTRACT Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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