An Atp-Dependent, Ran-Independent Mechanism for Nuclear Import of the U1a and U2b′′ Spliceosome Proteins

Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B′′ is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B′′ in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-β family of nucleocytoplasmic transport receptors, U1A/U2B′′ import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B′′ import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B′′ import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B′′ import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl2. These data suggest that U1A and U2B′′ import into the nucleus occurs by a hitherto uncharacterized mechanism.

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Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6337-6349 ◽

Cited By ~ 27

Author(s):

S E Wells ◽

M Ares

Keyword(s):

Synthetic Lethality ◽

Small Nuclear Rna ◽

Rna Structures ◽

Stem Loop ◽

U2 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

Nuclear Rna ◽

Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

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Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Molecular Endocrinology ◽

10.1210/me.2004-0334 ◽

2005 ◽

Vol 19 (7) ◽

pp. 1884-1892 ◽

Cited By ~ 39

Author(s):

Helena Sim ◽

Kieran Rimmer ◽

Sabine Kelly ◽

Louisa M. Ludbrook ◽

Andrew H. A. Clayton ◽

...

Keyword(s):

Y Chromosome ◽

Nuclear Import ◽

High Mobility ◽

Sex Reversal ◽

High Mobility Group ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

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Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4479-4487.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4479-4487

Author(s):

M Cotten ◽

G Schaffner ◽

M L Birnstiel

Keyword(s):

Antisense Rna ◽

Nuclear Extract ◽

Mrna Processing ◽

Rna Molecules ◽

Small Nuclear Ribonucleoprotein ◽

In Vitro Processing ◽

Antisense Dna ◽

Nuclear Ribonucleoprotein ◽

Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

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Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.495 ◽

1987 ◽

Vol 7 (1) ◽

pp. 495-503 ◽

Cited By ~ 18

Author(s):

L C Ryner ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Nuclear Extract ◽

Structural Features ◽

Simian Virus ◽

Micrococcal Nuclease ◽

Small Nuclear Ribonucleoprotein ◽

Cleavage And Polyadenylation ◽

Nuclear Ribonucleoprotein ◽

Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

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PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3710-3719.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3710-3719

Author(s):

J Banroques ◽

J N Abelson

Keyword(s):

Essential Role ◽

Mrna Splicing ◽

Ribonucleoprotein Particle ◽

Specific Antibodies ◽

Small Nuclear Ribonucleoprotein ◽

Assembly Pathway ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

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Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.495-503.1987 ◽

1987 ◽

Vol 7 (1) ◽

pp. 495-503

Author(s):

L C Ryner ◽

J L Manley

Keyword(s):

Simian Virus 40 ◽

Nuclear Extract ◽

Structural Features ◽

Simian Virus ◽

Micrococcal Nuclease ◽

Small Nuclear Ribonucleoprotein ◽

Cleavage And Polyadenylation ◽

Nuclear Ribonucleoprotein ◽

Cell Nuclear Extract

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Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1258-1269.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1258-1269

Author(s):

M Himmelspach ◽

R Gattoni ◽

C Gerst ◽

K Chebli ◽

J Stévenin

Keyword(s):

Donor Site ◽

Ribonucleoprotein Particle ◽

Adenovirus E1a ◽

Branch Site ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

The Common ◽

Nuclear Ribonucleoprotein

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

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005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

Reproduction Fertility and Development ◽

10.1071/srb05abs005 ◽

2005 ◽

Vol 17 (9) ◽

pp. 64

Author(s):

D. A. Jans ◽

G. Kaur ◽

I. K. H. Poon ◽

A. Delluc-Clavieries ◽

K. M. Wagstaff

Keyword(s):

Sex Determination ◽

Nuclear Localization ◽

Nuclear Import ◽

Sex Reversal ◽

Nuclear Accumulation ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Testicular Cells ◽

Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

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Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.703 ◽

2000 ◽

Vol 11 (2) ◽

pp. 703-719 ◽

Cited By ~ 26

Author(s):

Susanne M. Steggerda ◽

Ben E. Black ◽

Bryce M. Paschal

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Translocation ◽

Living Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Permeabilized Cells ◽

Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

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The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1210 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1210-1217 ◽

Cited By ~ 274

Author(s):

Ray Truant ◽

Bryan R. Cullen

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signals ◽

Immunodeficiency Virus ◽

Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

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