Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.     

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.     

p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly

The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m⁷G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.     

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.     

Climbing the vertebrate branch of U1A/U2B″ protein evolution

In the vertebrate lineage of the U1A/U2B″/SNF protein family, the U1A and U2B″ proteins bind to RNA stem–loops in the U1 or U2 snRNPs, respectively. However, their specialization is fairly recent, as they evolved from a single ancestral protein. The progress of their specialization (subfunctionalization) can be monitored by the amino acid sequence changes that give rise to their modern RNA-binding specificity. Using ancestral sequence reconstruction to predict the intermediates on the evolutionary branch, a probable path of sequential changes is defined for U1A and U2B″. The RNA-binding affinity for U1A/U2B″ protein ancestors was measured using modern U1 and U2 snRNA stem–loops and RNA stem–loop variants to understand how the proteins’ RNA specificities evolved.     

Xenopus LSm proteins bind U8 snoRNA via an internal evolutionarily conserved octamer sequence

U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.     

PSF and p54nrb bind a conserved stem in U5 snRNA

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.     

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.     

Drosophila SNF/D25 combines the functions of the two snRNP proteins U1A and U2B' that are encoded separately in human, potato, and yeast.

The plant and vertebrate snRP proteins U1A and U2B' are structurally closely related, but bind to different U snRNAs. Two additional related snRNP proteins, the yeast U2B' protein and Drosophila SNF/D25 protein, are analyzed here. We show that the previously described yeast open reading frame YIB9w encodes yeast U2B' as judged by the fact that the protein encoded by YIB9w bindsto stem-loop IV of yeast U2 snRNA in vitro and is part of the U2 snRNP in vivo. In contrast to the human U2B' protein, specific binding of yeast U2B' to RNA in vitro can occur in the absence of an accessory U2A' protein. The Drosophila SNF-D25 protein, unlike all other U1A/U2B' proteins studied to date, is shown to be a component of both U1 and U2 snRNPs. In vitro, SNF/D25 binds to U1 snRNA on itsown and to U2 snRNA in the presence of either the human U2A' protein or of Drosophila nuclear extract. Thus, its RNA-binding properties are the sum of those exhibited by human or potato U1A and U2B' proteins. Implications for the role of SNF/D25 in alternative splicing, and for the evolution of the U1A/U2B' protein family, are discussed.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

Evolutionary conservation of minor U12-type spliceosome between plants and humans

Splicing of rare, U12-type or AT-AC introns is mediated by a distinct spliceosome that assembles from U11, U12, U4atac, U6atac, and U5 snRNPs. Although in human cells the protein composition of minor and major snRNPs is similar, differences, particularly in U11 and U12 snRNPs, have been recently described. We have identified an Arabidopsis U11 snRNP-specific 35K protein as an interacting partner of an RS-domain-containing cyclophilin. By using a transient expression system in Arabidopsis protoplasts, we show that the 35K protein incorporates into snRNP. Oligo affinity selection and glycerol gradient centrifugation revealed that the Arabidopsis 35K protein is present in monomeric U11 snRNP and in U11/U12-di snRNP. The interaction of the 35K protein with Arabidopsis SR proteins together with its strong sequence similarity to U1-70K suggests that its function in splicing of minor introns is analogous to that of U1-70K. Analysis of Arabidopsis and Oryza sativa genome sequences revealed that all U11/U12-di-snRNP-specific proteins are conserved in dicot and monocot plants. In addition, we have identified an Arabidopsis gene encoding the homolog of U4atac snRNA and a second Arabidopsis gene encoding U6atac snRNA. Secondary structure predictions indicate that the Arabidopsis U4atac is able to form dimeric complexes with both Arabidopsis U6atac snRNAs. As revealed by RNaseA/T1 protection assay, the U4atac snRNA gene is expressed as an ~160-nt RNA, whereas the second U6atac snRNA gene seems to be a pseudogene. Taken together, our data indicate that recognition and splicing of minor, AT-AC introns in plants is highly similar to that in humans.     

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

Human U2B″ protein binding to snRNA stemloops

The human U2B″ protein is one of the unique proteins that comprise the U2 snRNP, but it is also a representative of the U1A/U2B″ protein family. In the U2 snRNP, it is bound to Stem-Loop IV (SLIV) of the U2 snRNA. We find that in vitro it binds not only to human SLIV, but also to Stem-Loop II (SLII) from human U1 snRNA and to Drosophila U2 snRNA SLIV. The thermodynamics of these binding interactions show a striking similarity, leading to the conclusion that U2B″ has a relaxed specificity for its RNA targets. The binding properties of U2B″ are distinct from those of human U1A and of Drosophila SNF, despite its high homology to those proteins, and so provide important new information on how this protein family has modulated its target preferences.