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Biomedical subjects

R Reed

Publications and source records attributed to R Reed.

At least 37 records · Page 2Linked to original sources
PubMed1999

Splicing is required for rapid and efficient mRNA export in metazoans.

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Deltai-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Deltai-mRNAs. Moreover, competition studies using excess Deltai-mRNA indicate that different factor(s) are involved in the inefficient export of Deltai-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Deltai-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Deltai-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.

Animals↗
PubMed1999

The RNA splicing factor hSlu7 is required for correct 3' splice-site choice.

The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.

Adenine↗
PubMed1999

Identification of both shared and distinct proteins in the major and minor spliceosomes.

In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the minor spliceosome and shed light on its evolutionary relationship to the major spliceosome.

Amino Acid Sequence↗
PubMed1999

Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing.

The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.

Adenoviridae↗
PubMed1999

Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking.

A variety of cellular processes are carried out by highly complex ribonucleoprotein (RNP) particles in which multiple RNA-RNA, RNA-protein, and protein-protein interactions occur. The spliceosome, which executes the nuclear pre-mRNA splicing reaction, is a particularly striking example of a complex RNP, containing a minimum of 50 distinct protein components as well as five small nuclear RNAs. In order to identify which among the numerous proteins may play critical roles in the splicing reaction, we have assembled spliceosomal complexes on pre-mRNA containing a single 32P-labeled nucleotide, isolated the complexes by gel filtration, and then carried out UV crosslinking. The combination of these three methods has allowed the identification of proteins that crosslink to critical sequence elements during each stage in spliceosome assembly. These methods should be generally applicable to the analysis of RNP complexes assembled in vitro.

Chromatography, Affinity↗
PubMed1999

Resolution of the mammalian E complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels.

A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

Adenosine Triphosphate↗
PubMed1999

Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155.

SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

Adaptor Proteins, Signal Transducing↗
PubMed1998

WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: the proline glycine and methionine-rich motif.

Pre-mRNA splicing requires the bridging of the 5' and 3' ends of the intron. In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP. Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP's), each of which contains one or more of a special class of tyrosine-rich WW domains. Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40. We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB', and the branchpoint binding protein SF1/mBBP. Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex.

Amino Acid Sequence↗
PubMed1998

Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis.

The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The role of stably committed and uncommitted cells in establishing tissues of the somite.

Somites are blocks of embryonic mesoderm tissue that give rise to skeletal muscle, cartilage, and other connective tissues. The development of different tissues within the somite is influenced by adjacent structures, in particular, the neural tube and notochord. Results of experiments performed in vivo and in vitro suggest that somites contain populations of cells stably programmed to undergo either skeletal myogenesis or chondrogenesis and a population uncommitted to either pathway. The fate of the uncommitted cells would depend on a transfer of information from the committed cells. Communication between committed and uncommitted cells is regulated by cell and tissue interactions that either activate or inhibit this process.

Animals↗
PubMed1998

Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing.

Pre-mRNA splicing takes place in two catalytic steps. The second step is poorly understood, especially in mammals. In yeast, the splicing factors, Prps 16, 17, 18 and Slu7 function exclusively in step II. Here we report the isolation of cDNAs encoding human Prps 16 and 17 which are 41 and 36% identical to their yeast counterparts. The Prp16 gene is essential in yeast, and we show that a chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout strain. Immunodepletion of hPrp16 from splicing extracts specifically blocks step II, and the activity can be fully restored with recombinant hPrp16. Moreover, both hPrps 16 and 17 associate with the spliceosome late in the splicing pathway. Mutations at the 3' splice site that specifically block step II do not affect the association of hPrps 16 and 17 with the spliceosome, indicating that these factors may function at a stage of step II prior to recognition of the 3' splice site. Recently, the human homologs of Prp18 and Slu7 were identified. The observation that humans contain homologs of all four known step II proteins in yeast indicates that the mechanism for catalytic step II is highly conserved.

Adenosine Triphosphatases↗
PubMed1998

Osteopontin stimulates a subpopulation of quiescent human prostate epithelial cells with high proliferative potential to divide in vitro.

BACKGROUND: Osteopontin (OPN) is a secreted extracellular matrix (ECM) protein found in bone, as well as associated with epithelial cells. The main objective of these studies was to test in vitro the hypothesis that interaction with OPN stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential. METHODS: To simulate conditions that restrict proliferation and inhibit terminal differentiation of basal cells in vivo, control cultures grew on substrate coated with collagen (CO) or fibronectin (FN), in medium containing low levels of growth factors. RESULTS: Under growth-restricting conditions, most prostate epithelial cells with high proliferative potential, seeded in control secondary cultures, were quiescent within the time frame of the studies, as indicated by the small number of large colonies in these cultures. Growing prostate epithelial cells (PR) under the same growth-restricting conditions, but on substrate coated with OPN instead of CO or FN, stimulated proliferation of a subpopulation of single cells with high proliferative ability as indicated by: 1) dose-dependent increase in the percentage of single cells incorporating bromodeoxyuridine, i.e., proliferating PR; and 2) subsequent dose-dependent increase in the percentage of large colonies. The OPN effect was not merely due to preferential attachment to OPN, because PR attachment to OPN, CO, or FN was identical. PR attachment to OPN was inhibited in the presence of GRGDTP or an antibody against the integrin subunit alphav, but not in the presence of an RGES peptide or a nonspecific IgG. CONCLUSIONS: Integrin-mediated OPN/PR interaction stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential, possibly stem cells.

Cell Adhesion↗
PubMed1998

Validating a measure of patient satisfaction with community nursing services.

This study describes an attempt to develop and validate a measure of patient satisfaction with a district nursing service in a south-west London inner city borough. The patients were 126 house-bound elderly people living alone and dependent upon district nursing services. The patient satisfaction measure was derived from an earlier North American questionnaire assessing satisfaction with hospital nursing services. The results indicated that the measure chosen reflected multiple dimensions of patient satisfaction that were differentially sensitive to factors such as the degree of personalization of care and the impact of disruptions to the service. Scores on the questionnaire were also related in a systematic way with the number and type of spontaneous comments made about the service. Overall the results suggest that self-report questionnaire measures of satisfaction can be devised that are sensitive to variations in the style and level of community nursing offered to older house-bound people.

Aged↗
PubMed1998

Association of guide RNA binding protein gBP21 with active RNA editing complexes in Trypanosoma brucei.

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

Animals↗
PubMed1998

Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells.

Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

Amino Acid Sequence↗