In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ϳ45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNAprocessing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. INTRODUCTIONAlthough eukaryotic ribosomes function in the cytoplasm, the synthesis, processing, and assembly of the ribosomal subunits in Saccharomyces cerevisiae and higher eukaryotes occur in the nucleolus. The entire ribosome is composed of four rRNA species and ϳ75 ribosomal proteins (r-proteins) distributed between two subunits. The 18S, 5.8S, and 25S rRNAs are derived from a single 35S rRNA precursor that is synthesized by RNA polymerase I and then processed by a series of endonucleolytic and exonucleolytic cleavages (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999). The 5S rRNA is synthesized separately by RNA polymerase III and associates with the 60S preribosomal subunit early in assembly. The mature 40S ribosomal subunit contains the 18S rRNA and ϳ32 r-proteins, whereas the 60S subunit is composed of the 5S, 5.8S, and 25S rRNAs and ϳ45 r-proteins. Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of r-proteins, the synthesis and processing of rRNA, and the concomitant assembly of r-proteins into the preribosomal subunits. Although a pathway for 35S rRNA maturation has been well defined through both genetic and biochemical approaches (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999), less is known about the association of r-proteins with the rRNA and the export of the assembled subunits out of the nucleus.All nucleocytoplasmic transport occurs ...

Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)10, as with L-phenylalanine and L-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a-d (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a > 1b > 1c > 1d, supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC50 potency: 1a/1b Ͼ Ͼ 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.biomaterial ͉ platelets ͉ structure-function ͉ supramolecular triplex T he self-association of peptides and proteins into well ordered supramolecular structures is of pivotal importance in normal physiology and pathophysiology, such as in the assembly of collagen fibrils (1), actin filaments (2), and amyloid fibrils (3, 4). Collagens, which constitute a ubiquitous protein family in animals, contribute an essential matrix component to soft tissues and bones (5, 6). A structural hallmark of many collagens is a rope-like triple helix, the architecture of which derives from the interplay of three proline-rich polypeptide strands (e.g., two ␣1 and one ␣2 for type I collagen) (6-8). In the core domain of the triple helix, the amino acid sequence G-X-Y is repeated multiple times, and each glycine amide NH forms a hydrogen bond with the X-position amide carbonyl on an adjacent strand. The X-and Y-positions are often populated by L-proline and 4(R)-hydroxy-L-proline (O; Hyp), respectively, with the latter stabilizing the triple helix by stereoelectronic effects (9) and water-bridged hydrogen bonds (10).To investigate collagen's structure and function, researchers have resorted to using synthetic collagen model peptides (CMPs)

A combination of several rate-limiting steps allows for efficient control of alternative splicing.

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.

SummaryNumerous non-coding RNAs are known to be involved in the regulation of gene expression. In this work, we analyzed RNAs that coimmunoprecipitated with human RNA polymerase II from mitotic cell extracts and identified U1 small nuclear RNA (snRNA) as a major species. To investigate a possible splicing-independent recruitment of U1 snRNA to transcription units, we established cell lines having integrated a reporter gene containing a functional intron or a splicing-deficient construction. Recruitment of U snRNAs and some splicing factors to transcription sites was evaluated using fluorescence in situ hybridization (FISH) and immunofluorescence. To analyze imaging data, we developed a quantitative procedure, 'radial analysis', based on averaging data from multiple fluorescence images. The major splicing snRNAs (U2, U4 and U6 snRNAs) as well as the U2AF65 and SC35 splicing factors were found to be recruited only to transcription units containing a functional intron. By contrast, U1 snRNA, the U1-70K (also known as snRNP70) U1-associated protein as well as the ASF/SF2 (also known as SFRS1) serine/arginine-rich (SR) protein were efficiently recruited both to normally spliced and splicing-deficient transcription units. The constitutive association of U1 small nuclear ribonucleoprotein (snRNP) with the transcription machinery might play a role in coupling transcription with pre-mRNA maturation.

The exon junction complex (EJC) allows the spliceosome to communicate with other cellular machinery. This study shows that assembled EJC cores are enriched in nuclear regions around speckles, called perispeckles. Speckles and perispeckles may represent specialized nuclear regions for messenger ribonucleoprotein maturation.

The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post-or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.

No abstract