The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex.

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.

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Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

Eukaryotic Cell ◽

10.1128/ec.00353-09 ◽

2010 ◽

Vol 9 (3) ◽

pp. 379-386 ◽

Cited By ~ 12

Author(s):

Nicolas Jaé ◽

Pingping Wang ◽

Tianpeng Gu ◽

Martin Hühn ◽

Zsofia Palfi ◽

...

Keyword(s):

Essential Role ◽

Core Protein ◽

Affinity Purification ◽

Rnase H ◽

Essential Step ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoproteins

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

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Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1578-1589.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1578-1589

Author(s):

L D Fresco ◽

D S Harper ◽

J D Keene

Keyword(s):

Protein Interactions ◽

Leucine Zipper ◽

Tandem Repeats ◽

Mutational Analysis ◽

Particle Density ◽

Protein Protein Interactions ◽

Small Nuclear Ribonucleoprotein ◽

Cell Extracts ◽

Associated Proteins ◽

Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

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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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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

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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Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.307 ◽

1993 ◽

Vol 13 (1) ◽

pp. 307-319 ◽

Cited By ~ 56

Author(s):

S E Behrens ◽

K Tyc ◽

B Kastner ◽

J Reichelt ◽

R Lührmann

Keyword(s):

Electron Microscope ◽

Salt Concentration ◽

Rnase H ◽

Globular Domain ◽

Branch Site ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

Specific Proteins ◽

Molecular Masses ◽

Nuclear Ribonucleoprotein

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.

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Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

Molecular and Cellular Biology ◽

10.1128/mcb.00560-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 5924-5936 ◽

Cited By ~ 73

Author(s):

AnYu Zhou ◽

Alexander C. Ou ◽

Aeri Cho ◽

Edward J. Benz ◽

Shu-Ching Huang

Keyword(s):

Splice Site ◽

Dependent Manner ◽

Splice Site Selection ◽

Exon 2 ◽

Small Nuclear Ribonucleoprotein ◽

U1 Snrnp ◽

Nuclear Ribonucleoprotein ◽

Dose Dependent ◽

Selection Of

ABSTRACT RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-xS 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-xL. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-xS 5′ ss selection, leading to decreased Bcl-xS isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-xS 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.

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U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3350 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3350-3359 ◽

Cited By ~ 49

Author(s):

D L Black ◽

A L Pinto

Keyword(s):

Rna Structure ◽

Rna Secondary Structure ◽

Rnase H ◽

Rna Sequences ◽

Large Loop ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Extracts ◽

The Individual ◽

Nuclear Ribonucleoprotein ◽

Dependent Interaction

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

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The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5543-5551.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5543-5551 ◽

Cited By ~ 131

Author(s):

Lili Wan ◽

Daniel J. Battle ◽

Jeongsik Yong ◽

Amelie K. Gubitz ◽

Stephen J. Kolb ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Motor Neurons ◽

Muscular Atrophy ◽

Protein Levels ◽

Small Nuclear Ribonucleoprotein ◽

Cell Extracts ◽

Smn Complex ◽

Survival Of Motor Neurons ◽

Smn Protein ◽

Nuclear Ribonucleoprotein

ABSTRACT Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

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