Nucleocytoplasmic transport and processing of small nuclear RNA precursors

We have analyzed the structures and locations of small nuclear RNA (snRNA) precursors at various stages in their synthesis and maturation. In the nuclei of pulse-labeled Xenopus laevis oocytes, we detected snRNAs that were longer than their mature forms at their 3' ends by up to 10 nucleotides. Analysis of the 5' caps of these RNAs and pulse-chase experiments showed that these nuclear snRNAs were precursors of the cytoplasmic pre-snRNAs that have been observed in the past. Synthesis of pre-snRNAs was not abolished by wheat germ agglutinin, which inhibits export of the pre-snRNAs from the nucleus, indicating that synthesis of these RNAs is not obligatorily coupled to their export. Newly synthesized U1 RNAs could be exported from the nucleus regardless of the length of the 3' extension, but pre-U1 RNAs that were elongated at their 3' ends by more than about 10 nucleotides were poor substrates for trimming in the cytoplasm. The structure at the 3' end was critical for subsequent transport of the RNA back to the nucleus. This requirement ensures that truncated and incompletely processed U1 RNAs are excluded from the nucleus.

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Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1553-1560.1992 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1553-1560

Author(s):

H Yang ◽

M L Moss ◽

E Lund ◽

J E Dahlberg

Keyword(s):

Xenopus Laevis ◽

Micrococcal Nuclease ◽

Xenopus Laevis Oocytes ◽

Small Nuclear Rna ◽

Trypsin Treatment ◽

Differential Processing ◽

Nuclear Extracts ◽

Nuclear Rna ◽

Processing Activity ◽

Nuclear Processing

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.

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Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1553 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1553-1560 ◽

Cited By ~ 17

Author(s):

H Yang ◽

M L Moss ◽

E Lund ◽

J E Dahlberg

Keyword(s):

Xenopus Laevis ◽

Micrococcal Nuclease ◽

Xenopus Laevis Oocytes ◽

Small Nuclear Rna ◽

Trypsin Treatment ◽

Differential Processing ◽

Nuclear Extracts ◽

Nuclear Rna ◽

Processing Activity ◽

Nuclear Processing

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.

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An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing

Journal of Cell Science ◽

10.1242/jcs.37.1.59 ◽

1979 ◽

Vol 37 (1) ◽

pp. 59-67

Author(s):

M. Geuskens ◽

R. Tencer

Keyword(s):

Wound Healing ◽

Plasma Membrane ◽

Electron Microscope ◽

Xenopus Laevis ◽

Ultrastructural Study ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Cell Cortex ◽

Animal Pole ◽

Annular Zone

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a ‘star’ around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

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Structural and functional analysis of chicken U4 small nuclear RNA genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3910 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3910-3919 ◽

Cited By ~ 12

Author(s):

M L Hoffman ◽

G M Korf ◽

K J McNamara ◽

W E Stumph

Keyword(s):

Distal Region ◽

Xenopus Laevis Oocytes ◽

Small Nuclear Rna ◽

Transcriptional Enhancer ◽

Base Pairs ◽

Base Substitutions ◽

Nuclear Rna ◽

Sequence Elements ◽

U4 Rna ◽

Rna Genes

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

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Cajal body surveillance of U snRNA export complex assembly

The Journal of Cell Biology ◽

10.1083/jcb.201004109 ◽

2010 ◽

Vol 190 (4) ◽

pp. 603-612 ◽

Cited By ~ 23

Author(s):

Tatsuya Suzuki ◽

Hiroto Izumi ◽

Mutsuhito Ohno

Keyword(s):

Xenopus Laevis ◽

Dominant Negative ◽

Cajal Body ◽

Cajal Bodies ◽

Xenopus Laevis Oocyte ◽

Small Nuclear Rna ◽

Body Surveillance ◽

Complex Assembly ◽

Nuclear Rna ◽

Rna Export

Phosphorylated adaptor for RNA export (PHAX) is the key export mediator for spliceosomal U small nuclear RNA (snRNA) precursors in metazoa. PHAX is enriched in Cajal bodies (CBs), nuclear subdomains involved in the biogenesis of small ribonucleoproteins. However, CBs’ role in U snRNA export has not been demonstrated. In this study, we show that U snRNA precursors microinjected into Xenopus laevis oocyte nuclei temporarily concentrate in CBs but gradually decrease as RNA export proceeds. Inhibition of PHAX activity by the coinjection of a specific anti-PHAX antibody or a dominant-negative PHAX mutant inhibits U snRNA export and simultaneously enhances accumulation of U snRNA precursors in CBs, indicating that U snRNAs transit through CBs before export and that binding to PHAX is required for efficient exit of U snRNAs from CBs. Similar results were obtained with U snRNAs transcribed from microinjected genes. These results reveal a novel function for CBs, which ensure that U snRNA precursors are properly bound by PHAX.

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Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus Laevis Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2419 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2419-2428 ◽

Cited By ~ 40

Author(s):

Thilo Sascha Lange ◽

Susan A. Gerbi

Keyword(s):

Nucleolar Localization ◽

Xenopus Laevis Oocytes ◽

Small Nuclear Rna ◽

U2 Snrna ◽

U6 Snrna ◽

Nuclear Rna ◽

Inxenopus Laevis ◽

Nucleolar Staining ◽

Over Time

Recent studies on the 2′-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

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An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. I. Inhibition of furrow formation

Journal of Cell Science ◽

10.1242/jcs.37.1.47 ◽

1979 ◽

Vol 37 (1) ◽

pp. 47-58

Author(s):

M. Geuskens ◽

R. Tencer

Keyword(s):

Plasma Membrane ◽

Xenopus Laevis ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Cytochalasin B ◽

Outer Side ◽

Cell Cortex ◽

Animal Pole ◽

Continuous Layer ◽

Close Proximity

Xenopus laevis fertilized eggs have been treated with wheat germ agglutinin (WGA) before the onset of the first cleavage, at the stripe stage and during groove deepening. The ultrastructure of the animal cortex of the arrested embryos has been compared with that of the same region of control embryos at different stages of first furrow formation and of cytochalasin B-treated embryos. The outer side of the plasma membrane of WGA-treated embryos is covered with a coat which is different from the diffuse material observed in either control or cytochalasin B-treated embryos and which is distributed in patches in the groove region. Narrow indentations of the plasma membrane in the cortex of WGA-treated eggs have been observed, particularly in the blocked or regressed groove. In WGA-treated eggs, a few bundles of microfilaments are located under the plasma membrane at the animal pole, but they are never arrayed in a continuous layer as in the control eggs. In the latter, many microtubules are located in close proximity to the microfilament layer at the beginning of cleavage, but they are only occasionally observed in the same region of WGA-treated eggs. It is concluded that the binding of WGA molecules to their receptors on the surface of the Xenopus zygote interferes with the alignment of microfilaments in the furrow region and provokes the disorganization of the aligned microfilaments once the cleavage has begun. Internalization of portions of the nascent membrane in the groove could play an important part in the arrest of cleavage.

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