Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells.
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The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells. (+info)
Synaptic plasticity: regulated translation in dendrites.
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Synaptic activity can induce neurons to synthesize proteins important for cognition and brain development. Recent results suggest this activity-induced protein synthesis is partially mediated by regulated translation within neuronal dendrites. (+info)
Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to histone pre-mRNA.
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The 3' end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3'-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3' processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP. (+info)
The suppressor of forked gene of Drosophila, which encodes a homologue of human CstF-77K involved in mRNA 3'-end processing, is required for progression through mitosis.
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The Suppressor of forked (Su(f)) protein of Drosophila melanogaster is a homologue of the 77K subunit of human cleavage stimulation factor required for cleavage of pre-mRNAs before addition of poly(A). We have previously shown that the Su(f) protein is not ubiquitously distributed: it accumulates in dividing cells at various stages of Drosophila development. In this paper, we show that phenotypes of su(f) temperature-sensitive mutants result from a defect in cell proliferation. Analysis of the mitotic phenotype of su(f) temperature-sensitive alleles in larval brain and in imaginal discs reveals an increase in the number of metaphases with overcondensed chromosomes and asymmetric or reduced mitotic spindles. In contrast, neural differentiation in eye imaginal discs of the same mutant flies does not appear to be affected. These results indicate that su(f) is required during cell division for progression through metaphase. Taken together, these data suggest that a decrease in su(f) activity preferentially affects 3'-end formation of particular mRNAs, some of which are involved in mitosis, and are in agreement with a role of su(f) in the regulation of poly(A) site utilization. (+info)
Two distinct forms of the 64,000 Mr protein of the cleavage stimulation factor are expressed in mouse male germ cells.
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Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3' ends but are efficiently polyadenylated. To determine whether the 64,000 Mr protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000 Mr form, we found a related approximately 70,000 Mr protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the approximately 70,000 Mr CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000 Mr form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000 Mr CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene. (+info)
Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly.
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We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor. (+info)
Assembly of the cleavage and polyadenylation apparatus requires about 10 seconds in vivo and is faster for strong than for weak poly(A) sites.
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We have devised a cis-antisense rescue assay of cleavage and polyadenylation to determine how long it takes the simian virus 40 (SV40) early poly(A) signal to commit itself to processing in vivo. An inverted copy of the poly(A) signal placed immediately downstream of the authentic one inhibited processing by means of sense-antisense duplex formation in the RNA. The antisense inhibition was gradually relieved when the inverted signal was moved increasing distances downstream, presumably because cleavage and polyadenylation occur before the polymerase reaches the antisense sequence. Antisense inhibition was unaffected when the inverted signal was moved upstream. Based on the known rate of transcription, we estimate that the cleavage-polyadenylation process takes between 10 and 20 s for the SV40 early poly(A) site to complete in vivo. Relief from inhibition occurred earlier for shorter antisense sequences than for longer ones. This indicates that a brief period of assembly is sufficient for the poly(A) signal to shield itself from a short (50- to 70-nucleotide) antisense sequence but that more assembly time is required for the signal to become immune to the longer ones (approximately 200 nucleotides). The simplest explanation for this target size effect is that the assembly process progressively sequesters more and more of the RNA surrounding the poly(A) signal up to a maximum of about 200 nucleotides, which we infer to be the domain of the mature apparatus. We compared strong and weak poly(A) sites. The SV40 late poly(A) site, one of the strongest, assembles several times faster than the weaker SV40 early or synthetic poly(A) site. (+info)
The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation.
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During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery. (+info)