An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Activation of a cryptic 5' splice site by U1 snRNA

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.     

Exon mutations uncouple 5' splice site selection from U1 snRNA pairing

It has previously been shown that a mutation of yeast 5' splice junctions at position 5 (GUAUGU) causes aberrant pre-mRNA cleavages near the correct 5' splice site. We show here that the addition of exon mutations to an aberrant cleavage site region transforms it into a functional 5' splice site both in vivo and in vitro. The aberrant mRNAs are translated in vivo. The results suggest that the highly conserved G at the 5' end of introns is necessary for the second step of splicing. Further analyses indicate that the location of the U1 snRNA-pre-mRNA pairing is not affected by the exon mutations and that the precise 5' splice site is selected independent of this pairing.     

Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

The association reaction of yeast alcohol dehydrogenase with coenzyme is partly diffusion-controlled in solvents of increased viscosity

The steady-state kinetics of the yeast and liver alcohol dehydrogenase catalyzed reduction of aldehydes were examined in solvent mixtures of increased viscosity. This was done to investigate the effects of diffusion control on the fast association of NADH with the enzymes. Both glycerol and sucrose were unsatisfactory as viscosogens, as they inhibited the enzyme, but poly(ethylene glycol)/water mixtures were satisfactory. The 5-fold faster reaction of yeast alcohol dehydrogenase with NADH is partly diffusion controlled, whereas the slower liver alcohol dehydrogenase reaction showed no diffusion effects. These results are consistent with a yeast alcohol dehydrogenase active site that has relatively little steric hindrance to NADH binding. It is estimated that contributions to this association reaction from diffusion control and chemical activation control are equal at a solvent viscosity of 10 cP. Thus, under physiological conditions of increased viscocity the NADH association may be significantly affected by diffusion effects. In order to estimate accurately the maximum diffusion-controlled rate constant from diffusion theory, the diffusion coefficients of NADH were measured in poly(ethylene glycol)/water mixtures and were found to vary inversely as the solvent viscosity raised to the power of 0.5. The non-Stokesian behaviour of molecules as large as NADH in polymer/water mixtures may be a serious limitation to the routine use of poly(ethylene glycol) as a viscosogen for diffusion studies.     

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.     

tau Exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.     

Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7-IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5' pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5' splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5' splice site, rather than repressing the 3' following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5' pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5' splice site follows a scanning-type mechanism, precluding competition with other functional 5' pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5' pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type-independent 5'-3'-oriented scanning process for accurate recognition of the authentic 5' splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5' splice site selection.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.     

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.     

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.     

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site.

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3'-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5'-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3'-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.