Pre-mRNA and mRNA binding of human nuclear DNA helicase II (RNA helicase A). (33/3744)

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, seems to function as a pre-mRNA and mRNA binding protein in human cells. Immunofluorescence studies of NDH II gave a highly diffused nucleoplasmic staining that was similar to that of hnRNP A1 but differed from the localization of the RNA splicing factor Sc-35. Upon transcriptional inhibition, NDH II migrated from the nucleus into the cytoplasm. During mitosis, NDH II was released into the cytoplasm during pro- to metaphase, and was gradually recruited back into telophase nuclei. The timing of nuclear import of NDH II at telophase was found to be later than that of hnRNP A1 but paralleled that of Sc-35. At the ultrastructural level, both NDH II and hnRNP A1 were identified within perichromatin ribonucleoparticle fibrils. However, the subnuclear distributions of NDH II and hnRNP A1 were not overlapping. NDH II could be extracted together with poly(A)-containing mRNA from HeLa cell nuclei and, to a much lesser extent, from the cytoplasm. Following transcriptional inhibition, NDH II was preferentially associated with mRNA from the cytosol, which biochemically confirmed the microscopic observations. Although NDH II is mainly a nuclear enzyme, it is apparently not associated with the nuclear matrix, since it could be extracted with 2 M NaCl from DNase I-treated nuclei. Our cellular and biochemical observations strongly suggest that NDH II is a pre-mRNA and mRNA binding protein. Its significant affinity for ssDNA, but not for dsDNA, points to a transient role in DNA binding during the process of transcript formation. According to our model, single-stranded DNA might be necessary to retain NDH II in the nuclear compartment.  (+info)

Coordination of tRNA nuclear export with processing of tRNA. (34/3744)

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.  (+info)

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA. (35/3744)

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing.  (+info)

Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF. (36/3744)

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, we have analyzed the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF). Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, we have identified Ser484 as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation.  (+info)

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization. (37/3744)

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.  (+info)

A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing. (38/3744)

The human papillomavirus (HPV) E2 protein plays an important role in transcriptional regulation of viral genes as well as in viral DNA replication. Unlike most types of HPV, the E2 protein of epidermodysplasia verruciformis (EV)-associated HPVs harbors a relatively long hinged region between the terminal, conserved transactivation and DNA binding/dimerization domains. The sequence of EV-HPV E2 hinge contains multiple arginine/serine (RS) dipeptide repeats which are characteristic of a family of pre-messenger RNA splicing factors, called SR proteins. Here we show that the HPV-5 (an EV-HPV) E2 protein can specifically interact with cellular splicing factors including a set of prototypical SR proteins and two snRNP-associated proteins. Transiently expressed HPV-5 E2 protein colocalizes with a nuclear matrix associated-splicing coactivator in nuclear speckled domains. The RS-rich hinge is essential for E2 transactivator interaction with splicing factors and for its subnuclear localization. Moreover, we present functional evidence for the HPV-5 E2 transactivator, which shows that the RS-rich hinge domain of the E2 protein can facilitate the splicing of precursor messenger RNA made via transactivation by E2 itself. Our results, therefore, suggest that a DNA binding transactivator containing an RS-rich sequence can play a dual role in gene expression.  (+info)

Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to histone pre-mRNA. (39/3744)

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)

RNA splicing: What has phosphorylation got to do with it? (40/3744)

Many pre-mRNA splicing factors are phosphorylated in vivo, but the role of this modification has been unclear. Recent observations suggest that phosphorylation modulates protein-protein interactions within the spliceosome, thereby contributing to dynamic structural reorganization of the spliceosome during splicing.  (+info)