Spliceosome assembly in the absence of stable U4/U6 RNA pairing

The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6–Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6–Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6–Prp24 complex renders stable U4/U6 pairing nonessential.     

Recycling of the U12-type spliceosome requires p110, a component of the U6atac snRNP

U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac snRNPs in addition to the U5 snRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 snRNPs during the spliceosome cycle, regenerating functional U4/U6 snRNPs from singular U4 and U6 snRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac snRNP. In contrast to the major U6 and U4/U6 snRNPs, p110 is primarily associated with the U6atac snRNP but is almost undetectable in the U4atac/U6atac snRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac snRNP.     

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

The uRNA database.

The uRNADB offers aligned, annotated and phylogenetically ordered sequences of several U RNAs. New to this release are RNAs from U7 (14 sequences), U8 (two sequences), U11 (three sequences), U12 (two sequences), U14 (11 sequences), U18, U48 and U49. A total of 34 new sequences were aligned with the previously compiled snRNAs U1, U2, U3, U4, U5 and U6.     

The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP.

Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

A novel U1/U5 interaction indicates proximity between U1 and U5 snRNAs during an early step of mRNA splicing.

The five spliceosomal snRNAs (U1, U2, U4, U5, and U6) undergo an ordered sequence of conformational changes as mRNA splicing progresses. We have shown that an antisense RNA oligonucleotide complementary to U5 snRNA induces a novel U1/U4/U5 complex that may be a transitional stage in the displacement of U1 from the 5' splice site by U5. Here we identify a novel site-specific crosslink between the 5' end of U1 and the invariant loop of U5 snRNA. This crosslink can be induced in nuclear extract by an antisense oligonucleotide directed against U5 snRNA, but can also be detected during an early step of the splicing reaction in the absence of oligonucleotide. Our data indicate proximity between U1 and U5 snRNPs before the first catalytic step of splicing, and may suggest that U1 helps to direct U5 to the 5' splice site.     

Hierarchical,clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.     

The Arabidopsis U12-type spliceosomal protein U11/U12-31K is involved in U12 intron splicing via RNA chaperone activity and affects plant development

U12 introns are removed from precursor-mRNA by a U12 intron-specific spliceosome that contains U11 and U12 small nuclear ribonucleoproteins. Although several proteins unique to the U12-type spliceosome have been identified, the manner by which they affect U12-dependent intron splicing as well as plant growth and development remain largely unknown. Here, we assessed the role of U11/U12-31K, a U12-type spliceosomal protein in Arabidopsis thaliana. T-DNA-tagged homozygote lines for U11/U12-31K could not be obtained, and heterozygote mutants were defective for seed maturation, indicating that U11/U12-31K is essential for the normal development of Arabidopsis. Knockdown of U11/U12-31K by artificial microRNA caused a defect in proper U12 intron splicing, resulting in abnormal stem growth and development of Arabidopsis. This defect in proper splicing was not restricted to specific U12-type introns, but most U12 intron splicing was influenced by U11/U12-31K. The stunted inflorescence stem growth was recovered by exogenously applied gibberellic acid (GA), but not by cytokinin, auxin, or brassinosteroid. GA metabolism-related genes were highly downregulated in U11/U12-31K knockdown plants. Importantly, U11/U12-31K was determined to harbor RNA chaperone activity. We propose that U11/U12-31K is an RNA chaperone that is indispensible for proper U12 intron splicing and for normal growth and development of plants.     

The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP.

We have raised specific antibodies against the PRP6 protein and shown that the U4, U5 and U6 snRNAs are co-precipitated with this protein. Using splicing extracts prepared from in vivo heat-inactivated cells, we have characterized the prp4-1 and prp6-1 biochemical defects. In inactivated prp4-1 cell extracts, the U6 snRNA content as well as the U6, U4/U6 snRNPs and the [U4/U6.U5] tri-snRNP particles amounts are severely reduced. In inactivated prp6-1 cell extracts, the PRP6 mutant protein is barely detectable. Glycerol gradient analyses indicate that, in these extracts, the [U4/U6.U5] tri-snRNPs are present in very low amounts, but U4/U6 snRNP particles are normally represented. These results establish that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. We found no evidence for the presence of the PRP6 protein in the U4/U6 particle.     

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

An in vitro reconstitution/splicing complementation system has been developed which has allowed the investigation of the role of mammalian U2 and U5 snRNP components in splicing. U2 or U5 snRNP cores are first reconstituted from purified native snRNP core proteins and snRNA in the absence of cellular extract and are subsequently added to splicing extracts depleted of either U2 or U5 snRNP. When snRNPs reconstituted with HeLa U2 or U5 snRNA were added to U2- or U5-depleted nuclear extract, splicing was complemented. Addition of naked snRNA, on the other hand, did not restore splicing, demonstrating that the core proteins are essential for both U2 and U5 snRNP functions in splicing. Hybrid U2 or U5 snRNPs, reconstituted with core proteins isolated from U1 or U2 snRNPs, were equally active in splicing complementation, indicating that the snRNP core proteins are functionally interchangeable. U5 snRNPs reconstituted from in vitro transcribed U5 snRNA restored splicing to a level identical to that observed with particles reconstituted from authentic HeLa U5 snRNA. In contrast, splicing could not be restored to U2-depleted extract by the addition of snRNPs reconstituted from synthetic U2 snRNA, suggesting that U2 snRNA base modifications are essential for U2 snRNP function.     

Systematics of the Usnea fragilescens aggregate and its distribution in Scandinavia

Systematics and taxonomy of Usnea cornuta, U. flammea and U. fragilescens are discussed. The morphology, chemistry and distribution of these species in Europe are described and a first attempt is made to provide a key for the erect-bushy to subpendant and sorediate species in Scandinavia. Compounds reported in this aggregate include lobaric acid (new to the genus). U. flammea is new for Scandinavia. U. constrictula, U. inflate, U. intexla and U. subpectinata are reduced to synonymy with U. cornuta, U. dalmatica and U. rupestris to synonymy with U. flammea, U. glaucescens to synonymy with U. hirta and the new combination U. fragilescens var. mollis is proposed.     

Susceptibility of 32 elm species and hybrids (Ulmus spp.) to the elm leaf beetle (Coleoptera: Chrysomelidae) under field conditions in Arizona

We evaluated elm leaf beetle, Pyrhalta luteola (Müller) (Coleoptera: Chrysomelidae), defoliation of 32 elm species or hybrids (taxa) established under field conditions in Holbrook, AZ. Percentage of defoliation, number of eggs, and number of larvae were estimated on randomly selected 15-cm shoot lengths annually in July, from 1996 to 2001. The following nine taxa consistently sustained 15-46% mean overall defoliation: 1) Siberian elm, U. pumila L.; 2) 'Dropmore' elm, U. pumila; 3) 'Camperdownii' elm, U. glabra Huds.; 4) 'Regal' elm, U. glabra x U. carpinifolia Gledisch x U. pumila); 5) 'Sapporo Autumn Gold' elm (U. pumila x U. japonica Sang.); 6) 'New Horizon' elm (U. pumila x U. japonica); 7) 'Charisma' elm [(U. japonica x U. wilsoniana Schneid.) x (U. japonica x U. pumila)]; 8) 'W2115-1' elm (U. parvifolia Jacq. x U. procera Salisb.); and 9) 'Homestead' elm [(U. hollandica Mill. x U. carpinifolia) x (U. pumila-racemosa Dieck x U. carpinifolia)]. Percentage of defoliation was significantly low on four Chinese elm (U. parvifolia) cultivars ('Allee', 'Athena', 'Glory'/lace bark, and 'Kings Choice'). Percentage of defoliation was also low on seven Asian elms (including U. chemnoui Cheng, U. bergmaniana Sneid., U. szechuanica Fang, and species of the U. davidiana Planch. complex [U. davidiana, U. japonica, U. wilsoniana, and U. propinqua Koidz.]) and the American elm (U. americana L.) 'Valley Forge'. Percentage of defoliation and the number of eggs or larvae per plant were highly correlated. The results of this study are generally consistent with results of past laboratory screening trials.     

A weak interaction between the U2A'' protein and U2 snRNA helps to stabilize their complex with the U2B" protein.

The U2 snRNP complex contains two specific proteins, U2B" and U2A'. We have analysed the interaction of U2A' with U2B" and with U2 RNA. U2A' can form an weak but detectable RNA-protein complex with U2 RNA and a stable protein complex with U2B". This protein-protein complex binds efficiently and specifically to U2 RNA. Binding experiments with mutant forms of U2A' shows that the region of U2A' essential for binding to U2B" is extensive, being located between amino acid position 1-164. The behaviour of the wild type U2A' protein, and in particular of a mutant version of the protein in which amino acids 3, 4 and 5 are mutated, suggests that U2A' forms a weak interaction with U2 RNA which helps to stabilize the U2A'-U2B"-U2 RNA complex. Mutants of U2 RNA were used to localize the region of U2 RNA important for interaction with U2A'. The results show that U2A' interacts with the stem of hairpin IV.     

Assembly and nuclear transport of the U4 and U4/U6 snRNPs

We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.     

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.     

Target discrimination by RNA-binding proteins: role of the ancillary protein U2A' and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B".

The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.