A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles

The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs). Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs. When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited. Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing. The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs.     

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.     

Four distinct regions in the auxiliary domain of heterogeneous nuclear ribonucleoprotein C-related proteins

The purpose of this presentation of pathological data is to demonstrate how our ideas have developed from a chronological point of view to our present interest in elementary cognitive processes. The present relative rarity of significant data in brain imagery, compared with what is observed in other fields of neuro-psychology, is probably due to the complexity of this cognitive processes.     

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.     

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.     

The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing

The splicing factor U2AF (U2 snRNP auxiliary factor) is a heterodimer with subunits of 65 and 35 kD (U2AF65 and U2AF35). U2AF65 binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF35. Here, we report that U2AF35 is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF35 were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF35. In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF35 directly mediates interactions between U2AF65 and proteins bound to the enhancers. Thus, U2AF35 functions as a bridge between U2AF65 and the enhancer complex to recruit U2AF65 to the adjacent intron.     

Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death

The U1 small nuclear ribonucleoprotein particle is essential for splicing of precursor mRNA, an activity that depends upon both the RNA and protein components of the U1 particle. One of the U1-specific proteins that is functionally important in this splicing reaction is the 70-kDa protein (U1-70kDa). We report here that U1-70kDa is specifically cleaved in apoptotic cells, resulting in the generation of a 40-kDa fragment. The kinetics of this cleavage coincided with the appearance of cells with apoptotic morphology in the population, and the proportion of 40-kDa fragment observed was markedly increased in apoptotic cells that had become detached from the substratum. Although the inhibitor characteristics of the activity cleaving U1-70kDa suggest that interleukin 1 beta-converting enzyme (ICE) might be responsible, the specific ICE inhibitor N-(N-acetyl-tyrosinyl-valinyl-alaninyl)-3-amino-4-oxob utanoic acid (YVAD-CHO) did not prevent cleavage, and U1-70kDa was not cleaved by purified ICE in vitro. Further study of this novel cleavage and the enzyme responsible will yield information about proteolytic events that might be central in the mechanism and control of apoptosis.     

Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA

We have determined the crystal structure at 2.4 A resolution of a ternary complex between the spliceosomal U2B"/U2A' protein complex and hairpin-loop IV of U2 small nuclear RNA. Unlike its close homologue the U1A protein, U2B" binds to its cognate RNA only in the presence of U2A', which contains leucine-rich repeats in its sequence. The concave surface of a parallel beta-sheet within the leucine-rich-repeat region of U2A' interacts with the ribonucleoprotein domain of U2B" on the surface opposite its RNA-binding surface. The basic carboxy-terminal region of U2A' interacts with the RNA stem. The crystal structure reveals how protein-protein interaction regulates RNA-binding specificity, and how replacing only a few key residues allows the U2B" and U1A proteins to discriminate between their cognate RNA hairpins by forming alternative networks of interactions.     

U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.     

RNA binding activity of heterodimeric splicing factor U2AF: at least one RS domain is required for high-affinity binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50DeltaRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

Discovery of insect anti-juvenile hormones in plants.?2U

Two simple chromenes with anti-JH activity have been isolated and identified from the bedding plant Ageratum houstoianum. By contact and fumigation these compounds induce precocious metamorphosis and sterilization in several hemipteran species of insects. Certain holometabolous species are sterilized, forced into diapause, or both. Each of these biological actions is equivalent to removal of the corpora allata, which produce the JH's, and is reversible by treatment with exogenous JH. Thus, the action of these compounds is to stop the production or depress the titer of the JH's. To our knowledge, this is the first discovery of anti-JH, and we hope it will guide the way to the emergence of a fourth generation of safe and insect-specific pesticides.     

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 in bronchial epithelium of chronic smokers

The monoclonal antibody 703D4, which binds heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), has been reported to detect lung cancer more than a year earlier than routine chest X-ray or cytomorphology. To explore the biological basis of this detection, we studied the expression of this antigen in the central airways of smokers with evidence of bronchial metaplasia using specimens from a previously reported, randomized retinoid chemoprevention trial. By analyzing 1078 available biopsy specimens from 147 individuals at baseline and 68 individuals who completed the intervention, we frequently detected overexpression of hnRNP A2/B1 in normal and abnormal bronchial epithelium (i.e., in 41% of normal and 37% of squamous metaplasia samples). There was no correlation between hnRNP A2/B1 overexpression and the different histological changes. In cases with hnRNP A2/B1 overexpression, immunoreactivity was homogeneously expressed in all biopsied sites. For the 68 cases with serial biopsies, there was no significant modulation of hnRNP expression by retinoid intervention or smoking status. With lung cancer cell lines, 0.5-4 microM concentrations of 13-cis-retinoic acid reduced hnRNP A2/B1 overexpression by immunocytochemistry. We conclude that hnRNP A2/B1 overexpression is frequently found in central airways of chronic smokers, consistent with the pattern of expression that we reported previously in airways surrounding resected primary lung cancers. Oral 13-cis-retinoic acid at a dose of 1 mg/kg has no demonstrable effects on modulating hnRNP A2/B1 expression in proximal bronchial epithelium.     

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.     

Interaction between the negative regulator of splicing element and a 3' splice site: requirement for U1 small nuclear ribonucleoprotein and the 3' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.