Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast. (+info)
Association of the rat heterogeneous nuclear RNA-ribonucleoprotein F with TATA-binding protein.
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Heterogeneous nuclear ribonucleoprotein F (hnRNP-F) has been shown to be a pre-mRNA splicing factor. Recent studies have uncovered the coordination of synthesis of pre-mRNA and its processing, including post-transcriptional modification and splicing. Here, we present evidence for an association between a splicing factor, hnRNP-F, and TATA-binding protein (TBP), which is an essential factor needed for transcription initiation. An affinity detection experiment revealed hnRNP-F in the preparation of TBP-interacting proteins. HnRNP-F was associated with TBP in nuclear extracts and was capable of direct binding to TBP in vitro. These results suggest that hnRNP-F is associated with TBP in the cell. HnRNP-F was observed in abundance in the thymus, spleen and testis, and its distribution pattern was similar to that of TBP, implying a functional coordination of transcription and splicing. We assume that the splicing machinery is associated with the transcription apparatus as a prerequisite prior to transcriptional elongation. (+info)
Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.
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The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin-hinge-hairpin-tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization. (+info)
Signal recognition particle components in the nucleolus.
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The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves. (+info)
Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA.
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The promyelocytic leukemia (PML) nuclear body (also referred to as ND10, POD, and Kr body) is involved in oncogenesis and viral infection. This subnuclear domain has been reported to be rich in RNA and a site of nascent RNA synthesis, implicating its direct involvement in the regulation of gene expression. We used an analytical transmission electron microscopic method to determine the structure and composition of PML nuclear bodies and the surrounding nucleoplasm. Electron spectroscopic imaging (ESI) demonstrates that the core of the PML nuclear body is a dense, protein-based structure, 250 nm in diameter, which does not contain detectable nucleic acid. Although PML nuclear bodies contain neither chromatin nor nascent RNA, newly synthesized RNA is associated with the periphery of the PML nuclear body, and is found within the chromatin-depleted region of the nucleoplasm immediately surrounding the core of the PML nuclear body. We further show that the RNA does not accumulate in the protein core of the structure. Our results dismiss the hypothesis that the PML nuclear body is a site of transcription, but support the model in which the PML nuclear body may contribute to the formation of a favorable nuclear environment for the expression of specific genes. (+info)
Half a century of "the nuclear matrix".
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A cell fraction that would today be termed "the nuclear matrix" was first described and patented in 1948 by Russian investigators. In 1974 this fraction was rediscovered and promoted as a fundamental organizing principle of eukaryotic gene expression. Yet, convincing evidence for this functional role of the nuclear matrix has been elusive and has recently been further challenged. What do we really know about the nonchromatin elements (if any) of internal nuclear structure? Are there objective reasons (as opposed to thinly veiled disdain) to question experiments that use harsh nuclear extraction steps and precipitation-prone conditions? Are the known biophysical properties of the nucleoplasm in vivo consistent with the existence of an extensive network of anastomosing filaments coursing dendritically throughout the interchromatin space? To what extent may the genome itself contribute information for its own quarternary structure in the interphase nucleus? These questions and recent work that bears on the mystique of the nuclear matrix are addressed in this essay. The degree to which gene expression literally depends on nonchromatin nuclear structure as a facilitating organizational format remains an intriguing but unsolved issue in eukaryotic cell biology, and considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the in vivo situation. (+info)
Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis.
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Human aminoacyl-tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl-tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm. (+info)
Length increase of the human alpha -globin 3'-untranslated region disrupts stability of the pre-mRNA but not that of the mature mRNA.
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Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we have found that alpha-globin pre-mRNA is stabilized by a mechanism that does not modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. We found an inverse relationship between mRNA levels and the length of the introduced fragments. In fact, mRNA levels as low as 1% were observed after inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment of 86 nt at the same position had no effect on mRNA accumulation. DNA insertion induced no change in transcription rate or in half-life of mature mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreased levels of nuclear pre-mRNA in proportion to that observed for mature mRNA. In contrast, the insertion of the 477-nt exogenous DNA in the last intron had no effect on mRNA levels despite the presence of intronic sequences in the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR. The fact that the half-life of mature mRNA is not affected by DNA insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation. (+info)