Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs.

Each step in the biogenesis of microRNAs (miRNAs) depends on recognition of a correct substrate and efficient transport or processing of that RNA. Exportin5 (Exp5) and Dicer are proteins that mediate two key steps in this cascade, the nuclear export and cytoplasmic processing of microRNA precursor (pre-miRNAs). Xenopus laevis oocytes, eggs, and embryos constitute convenient experimental systems in which to study the substrate specificity of these proteins because specific RNAs or proteins can be injected directly into different subcellular compartments. We have used the Xenopus system and in vitro processing to define and compare the specificities of Exp5 and Dicer. Although both proteins act on many of the same substrates, we show that they recognize different structure elements of these RNAs. Our studies also revealed several unexpected activities. For example, Exp5 can mediate export of unspliced pre-mRNAs and excised lariat introns if these RNAs contain an aptamer sequence that itself is an Exp5 export substrate. Finally, we demonstrate that maturation of Xenopus oocytes into eggs leads to a large increase in Dicer activity, suggesting that miRNA biogenesis is subject to developmental control.

Economic consequences of an implanted neuroprosthesis for bladder and bowel management.

OBJECTIVE: To determine whether an implanted neuroprosthesis for bladder and bowel management is less costly than conventional techniques. DESIGN: Retrospective cost-identification analysis with comparison before and after implantation of the neuroprosthesis. SETTING: Life-care planning interviews in patients' homes. PATIENTS: Twelve patients with complete suprasacral spinal cord injuries and neurogenic bladder and bowel. INTERVENTION: Implantation of a neuroprosthesis for electric stimulation of the sacral nerves and posterior sacral rhizotomy. MAIN OUTCOME MEASURES: Annual costs of bladder and bowel care with and without the neuroprosthesis, projected over 10 years. RESULTS: Bladder and bowel care costs were reduced by over 80%, from a median of 8152 dollars a year for conventional care to a median of 948 dollars a year. With the neuroprosthesis, median annual costs for bladder supplies were reduced from 3368 dollars to 58 dollars; for medications, from 1866 dollars to 108 dollars; for medical care, from 656 dollars to 96 dollars; and for bowel care supplies, from 205 dollars to 87 dollars. After 5 years, the cumulative costs of treatment with the neuroprosthesis, including the cost of the device and its implantation and maintenance, equaled those of conventional care. Thereafter, savings from the implanted neuroprosthesis are projected to increase progressively throughout the patient's life. CONCLUSION: A neuroprosthesis implant with posterior rhizotomy greatly reduces the cost of managing the neurogenic bladder and bowel.

Multiple vesiculoviral matrix proteins inhibit both nuclear export and import.

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.

Reduction of costs of disability using neuroprostheses.

The lifetime costs associated with spinal cord injury are substantial. Assistive technology that reduces complications, increases independence, or decreases the need for attendant services can provide economic as well as medical or functional benefit. This study describes two approaches for estimating the economic consequences of implanted neuroprostheses utilizing functional electrical stimulation. Life care plan analysis was used to estimate the costs of bladder and bowel care with and without a device restoring bladder and bowel function and to compare these with the costs of implementing the device. For a neuroprosthesis restoring hand grasp, the costs of implementation were compared to the potential savings in attendant care costs that could be achieved by the use of the device. The results indicate that the costs of implementing the bladder and bowel system would be recovered in 5 years, primarily from reduced costs of supplies, medications, and procedures. The costs of the hand grasp neuroprosthesis would be recovered over the lifetime of the user if attendant time was reduced only 2 hours per day and in a shorter time if attendant care was further reduced. Neither analysis includes valuation of the quality of life, which is further enhanced by the neuroprostheses through restoration of greater independence and dignity. Our results demonstrate that implantable neuroprosthetic systems provide good health care value in addition to improved independence for the disabled individual.

The matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of bidirectional nuclear transport. Here we demonstrate that inhibition occurs when M protein is in the nucleus of Xenopus laevis oocytes and that M activity is readily reversed by a monoclonal antibody (alphaM). We identify a region of M protein, amino acids 51 to 59, that is required both for inhibition of transport and for efficient recognition by alphaM. When expressed in transfected HeLa cells, M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim. Moreover, mutation of a single amino acid, methionine 51, eliminates both transport inhibition and targeting to NPCs. We propose that M protein inhibits bidirectional transport by interacting with a component of the NPC or an NPC-associated factor that participates in nucleocytoplasmic transport.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Comparison of the 5' nuclease activities of taq DNA polymerase and its isolated nuclease domain.

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5' nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5' arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5' nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3' arm of the bifurcation; optimal stimulation requires overlap of the 3' terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.

Proofreading and aminoacylation of tRNAs before export from the nucleus.

After synthesis and processing in the nucleus, mature transfer RNAs (tRNAs) are exported to the cytoplasm in a Ran.guanosine triphosphate-dependent manner. Export of defective or immature tRNAs is avoided by monitoring both structure and function of tRNAs in the nucleus, and only tRNAs with mature 5' and 3' ends are exported. All tRNAs examined can be aminoacylated in nuclei of Xenopus oocytes, thereby providing a possible mechanism for functional proofreading of newly made tRNAs. Inhibition of aminoacylation of a specific tRNA retards its appearance in the cytoplasm, indicating that nuclear aminoacylation promotes efficient export.

Functions of the GTPase Ran in RNA export from the nucleus.

Significant and exciting advances in the field of RNA and protein export have been made recently, due in large part to discovery of the roles played by Ran, a small, soluble GTPase present in both the nucleus and cytoplasm of all eukaryotic cells. Ran is thought to be primarily bound to GTP in the nucleus and to GDP in the cytoplasm, as a result of the assymetric distribution of factors that interact with Ran to promote guanine nucleotide exchange (in the nucleus) and GTP hydrolysis (in the cytoplasm). A key function of the nuclear Ran.GTP is to support formation of complexes containing an export receptor (an exportin) and cargos such as RNAs, RNPs or proteins that are destined for export. In the cytoplasm, removal of the Ran.GTP from the complex results in its destabilization and release of the export cargo. Although Ran.GTP is required for formation of the export complex, GTP hydrolysis does not appear to be necessary for translocation through the nuclear pore complex or cytoplasmic release. Nevertheless, the GTPase of Ran does appear to be required in as yet unidentified intranuclear steps prior to export of some, but not all, RNAs.

Selection and nuclear immobilization of exportable RNAs.

The intracellular distribution of RNAs depends on interactions of cis-acting nuclear export elements or nuclear retention elements with trans-acting nuclear transport or retention factors. To learn about the relationship between export and retention, we isolated RNAs that are exported from nuclei of Xenopus laevis oocytes even when most RNA export is blocked by an inhibitor of Ran-dependent nucleocytoplasmic transport, the Matrix protein of vesicular stomatitis virus. Export of the selected RNAs is saturable and specific. When present in chimeric RNAs, the selected sequences acted like nuclear export elements in promoting efficient export of RNAs that otherwise are not exported; the pathway used for export of these chimeric RNAs is that used for the selected RNAs alone. However, these chimeric RNAs, unlike the selected RNAs, were not exported in the presence of Matrix protein; thus, the nonselected sequences can cause retention of the selected RNA sequences under conditions of impaired nucleocytoplasmic transport. We propose that most RNAs are transiently immobilized in the nucleus and that release of these RNAs is an essential and early step in export. Release correlates with functional Ran-dependent transport, and the lack of export of chimeric RNAs may result from interference with the Ran system.

Inhibition of Ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus.

Transport of macromolecules into and out of nuclei, essential steps in gene expression, are potential points of control. The matrix protein (M protein) of vesicular stomatitis virus (VSV) was shown to block transport of RNAs and proteins between the nucleus and cytoplasm of Xenopus laevis oocytes. The pattern of inhibition indicated that M protein interfered with transport that is dependent on the ras-like nuclear guanosine triphosphatase (GTPase) Ran-TC4 and its associated factors. This inhibition of nuclear transport by M protein explains several observations about the effects of VSV infection on host cell gene expression and suggests that RNA export is closely coupled to protein import.

Control of mouse U1 snRNA gene expression during in vitro differentiation of mouse embryonic stem cells.

Early in mouse development, two classes of U1 RNAs, mU1a and mU1b, are synthesized, but as development proceeds, transcription of the embryo-specific mU1b genes is selectively down-regulated to a barely detectable level. We show here that during in vitro differentiation of mouse embryonic stem (ES) cells, both exogenously introduced and endogenous U1b genes are subject to normal developmental regulation. Thus, ES cells represent a convenient isogenic system for studying the control of expression of developmentally regulated snRNA genes. Using this system, we have identified a region in the proximal 5'flanking region, located outside the PSE element, that is responsible for differential transcription of the mU1a and mU1b genes in both developing cells and transiently transfected NIH 3T3 cells.

In vivo selection of RNAs that localize in the nucleus.

Nuclear localization of an RNA is affected by cis-acting elements (NLEs) that lead to nuclear import or retention or to blockage of export from the nucleus. To identify such elements, we selected and analyzed transcripts that localized in the nuclei of Xenopus laevis oocytes. The RNAs were isolated from a collection of m7G-capped RNAs in which a combinatorial library (n = 20) of sequences had been inserted. One class of selected RNAs (Sm+) had a consensus Sm binding site (AAUUUUUGG) and bound Sm proteins in the cytoplasm; these RNAs resembled small nuclear RNAs like U1 and U5 RNAs in their bi-directional nucleocytoplasmic transport and their 5'-cap hypermethylation. Another class, Sm- RNAs, contained sequences that masked the m7G-caps of the RNAs and promoted interaction with La protein. These RNAs were retained within nuclei after nuclear injection and were imported when injected into the cytoplasm. Their nuclear import and retention were independent of a 5'-cap, required an imperfect double-stranded stem near the 5' end, and depended on interaction with La protein. Import of the Sm- RNAs, while using the import pathway of proteins, was distinct from that of U6 RNA.

Coupling of nuclear RNA export and protein import in vertebrate cells.

Nuclear RNA export is a highly dynamic process in which factors that carry the RNAs out of the nucleus must be re-imported. These RNA export factors are bifunctional molecules that recognize specific RNA sequences or structures and interact with shared nuclear proteins, including components of the nuclear pore complex. Inhibition of protein import by inactivation of the GTPase Ran, or its associated activating and recycling factors, blocks RNA export. However a few classes of RNAs escape this inhibition, perhaps because they do not use shuttling export factors or their factors have been stockpiled in the nucleus. Because of its critical role in gene expression, RNA export is a target for control by both cells and viruses.

The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways.

The 97-kD O-linked glycoprotein, Nup98, is a component of the Xenopus laevis nuclear pore complex and the only vertebrate GLFG nucleoporin identified (Powers, M.A., C. Macauley, F. Masiarz, and D.J. Forbes. 1995. J. Cell Biol. 128:721-736). We have investigated possible roles of xNup98 in the nucleocytoplasmic transport of proteins and RNAs by analyzing the consequences of injecting monospecific polyclonal antibodies to xNup98 into X. laevis oocytes. We show here that nuclear injection of anti-xNup98 inhibited the export of multiple classes of RNAs, including snRNAs, 5S RNA, large ribosomal RNAs, and mRNA. In contrast, the export of tRNA was unaffected. Injection of anti-xNup98 into the oocyte cytoplasm had no effect on export of any of the RNAs. Significantly, nuclear injection of anti-xNup98 antibodies did not inhibit import of either karyophilic proteins or snRNPs. This latter result is in agreement with our previous finding that Nup98 is not an essential element of the protein import pathway. Thus, Nup98 plays a role specifically in RNA export from the nucleus, and it appears to be an essential component of multiple RNA export pathways.

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.

The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates.

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10-20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage.

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.

Reverse 5' caps in RNAs made in vitro by phage RNA polymerases.

We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.