The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.     

The human U6 snRNA intramolecular helix: structural constraints and lack of sequence specificity.

Splicing of mRNA precursors occurs in a massive structure known as the spliceosome and requires the function of several small nuclear RNAs (snRNAs). A number of studies have suggested potentially important roles for two snRNAs, U2 and U6, in splicing catalysis. These two RNAs interact extensively with each other, as well as with the pre-mRNA, and possible similarities with catalytic RNAs have been noted. An important feature of the U2-U6 complex is an intramolecular helix in U6, which forms in conjunction with activation of the spliceosome. Here we describe a detailed genetic analysis of residues that make up this helix in human U6 snRNA, using an in vivo assay in which splicing of a test pre-mRNA is dependent on exogenous U6 snRNA. Our results show that many, but not all, positions tested are sensitive to mutation. Unexpectedly, base pairing is fully compatible with function at all positions, and at many is both necessary and sufficient. For example, conversion of two noncanonical A-C pairs to G-C pairs did not affect splicing, nor did conversion of an A-G to C-G. Extension of the helix by a base pair was also tolerated, provided that base pairing was maintained. Most notable was the behavior of a bulged U (U74), which has been suggested previously to be of particular importance. Although U74 was sensitive to substitution or deletion, incorporation into the helix by insertion of an A across from it was without effect, even in the context of a second helix-stabilizing mutation. We discuss these results in terms of possible mechanisms by which U6 snRNA might function in splicing catalysis.     

Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase.

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.     

Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.

U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly.     

Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA

Using a combination of RNA sequencing and construction of cDNA clones followed by DNA sequencing, we have determined the primary nucleotide sequence of U3 snRNA in Xenopus laevis and Xenopus borealis. This molecule has a length of 219 nucleotides. Alignment of the Xenopus sequences with U3 snRNA sequences from other organisms reveals three evolutionarily conserved blocks. We have probed the secondary structure of U3 snRNA in intact Xenopus laevis nuclei using single-strand specific chemical reagents; primer extension was used to map the positions of chemical modification. The three blocks of conserved sequences fall within single-stranded regions, and are therefore accessible for interaction with other molecules. Models of U3 snRNA function are discussed in light of these data.     

Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model.

The solution structure of human U1 snRNA was investigated by using base-specific chemical probes (dimethylsulfate, carbodiimide, diethylpyrocarbonate) and RNase V1. Chemical reagents were employed under various conditions of salt and temperature and allowed information at the Watson-Crick base-pairing positions to be obtained for 66% of the U1 snRNA bases. Double-stranded or stacked regions were examined with RNase V1. The dat gained from these experiments extend and support the previous 2D model for U1snRNA. However, to elucidate some aspects of the solution data that could not be accounted for by the secondary structure model, the information gathered from structure probing was used to provide the experimental basis required to construct and to test a tertiary structure model by computer graphics modeling. As a result, U1 snRNA is shown to adopt an asymmetrical X-shape that is formed by two helical domains, each one being generated by coaxial stacking of helices at the U1 snRNA cruciform. Chemical reactivities and model building show that a few nucleotides, previously proposed to be unpaired, can form A.G and U.U non Watson-Crick base-pairs, notably in stem-loop B. The structural model we propose for regions G12 to A124 integrates stereochemical constraints and is based both on solution structure data and sequence comparisons between U1 snRNAs.     

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

Chemical modification of cytosine residues of mouse 5 S ribosomal RNA with hydrogen sulfide (Nucleosides and nucleotides 43)

Cytosine residues of nucleic acids were converted to 4-thiouracil residues with hydrogen sulfide in pyridine and water to examine the secondary and tertiary structures of mouse 5 S rRNA. The cytosine residues at positions 10, 24, 34 (or 36), 39, 44 (or 46) and 63 were converted preferentially when the treatment was carried out at 28°C. This result supports the model of the secondary structure of 5 S rRNA of Nishikawa, K. and Takemura, S. ((1974) FEBS Lett. 40, 106–109) consisting of five helices and five loops. As the temperature was increased to 35°C, additional cytosine residues in positions 26, 52 and 78 were modified to moderate extents.     

A phylogenetic study of U4 snRNA reveals the existence of an evolutionarily conserved secondary structure corresponding to 'free' U4 snRNA

The nucleotide sequence of Physarum polycephalum U4 snRNA*** was determined and compared to published U4 snRNA sequences. The primary structure of P polycephalum U4 snRNA is closer to that of plants and animals than to that of fungi. But, both fungi and P polycephalum U4 snRNAs are missing the 3' terminal hairpin and this may be a common feature of lower eucaryote U4 snRNAs. We found that the secondary structure model we previously proposed for 'free' U4 snRNA is compatible with the various U4 snRNA sequences published. The possibility to form this tetrahelix structure is preserved by several compensatory base substitutions and by compensatory nucleotide insertions and deletions. According to this finding, association between U4 and U6 snRNAs implies the disruption of 2 internal helical structures of U4 snRNA. One has a very low free energy, but the other, which represents one-half of the helical region of the 5' hairpin, requires 4 to 5 kcal to be open. The remaining part of the 5' hairpin is maintained in the U4/U6 complex and we observed the conservation, in all U4 snRNAs studied, of a U bulge residue at the limit between the helical region which has to be melted and that which is maintained. The 3' domain of U4 snRNA is less conserved in both size and primary structure than the 5' domain; its structure is also more compact in the RNA in solution. In this domain, only the Sm binding site and the presence of a bulge nucleotide in the hairpin on the 5' side of the Sm site are conserved throughout evolution.     

Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants

Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence.     

Mammalian U6 small nuclear RNA undergoes 3'' end modifications within the spliceosome.

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.     

Proposed secondary structure of eukaryotic U14 snRNA.

U14 snRNA is a small nuclear RNA that plays a role in the processing of eukaryotic ribosomal RNA. We have investigated the folded structure of this snRNA species using comparative analysis of evolutionarily diverse U14 snRNA primary sequences coupled with nuclease digestion analysis of mouse U14 snRNA. Covariant nucleotide analysis of aligned mouse, rat, human, and yeast U14 snRNA primary sequences suggested a basic folding pattern in which the 5' and 3' termini of all U14 snRNAs were base-paired. Subsequent digestion of mouse U14 snRNA with mung bean (single-strand-specific), T2 (single-strand-preferential), and V1 (double-strand-specific) nucleases defined the major and minor cleavage sites for each nuclease. This digestion data was then utilized in concert with the comparative sequence analysis of aligned U14 snRNA primary sequences to refine the secondary structure model suggested by computer-predicted folding. The proposed secondary structure of U14 snRNA is comprised of three major hairpin/helical regions which includes the helix of base-paired 5' and 3' termini. Strict and semiconservative covariation of specific base-pairs within two of the three major helices, as well as nucleotide changes that strengthen or extend base-paired regions, support this folded conformation as the evolutionary conserved secondary structure for U14 snRNA.     

Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP

We describe the electron microscopic investigation of purified U4/U6 snRNPs from human and murine cells. The U4/U6 snRNP exhibits two morphological features, a main body approximately 8 nm in diameter and a peripheral filamentous domain, 7-10 nm long. Two lines of evidence suggest that the peripheral domain may consist of RNA and to contain U6 RNA as well as the 5' portion of U4 RNA. (a) Separation of the U4/U6 snRNA interaction regions from the core domains by site-directed cleavage of the U4 snRNA with RNase H gave filament-free, globular core snRNP structures. (b) By immuno and DNA-hybridization EM, both the 5' end of U4 and the 3' end of U6 snRNA were located at the distal region of the filamentous domain, furthest from the core. These results, together with our observation that the filamentous U4/U6 domain is often Y shaped, correlate strikingly with the consensus secondary structure proposed by Brow and Guthrie (1988. Nature (Lond.), 334:213-218), where U4 and U6 snRNA are base paired in such a way that two U4/U6 helices together with a stem/loop of U4 snRNA make up a Y-shaped U4/U6 interaction domain.     

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Recognition of a bulged RNA by peptides derived from the influenza NS1 protein

A competition assay for RNA binding by the influenza virus NS1 protein using model RNAs, U6-45, corresponding to U6 snRNA revealed that deletion of each of the three bulged-out parts reduced the NS1 protein binding and, in contrast, by deleting all three of the bulged-out parts, simultaneously, and thus producing a double-stranded RNA, the binding was recovered. A common feature of target RNAs of the NS1 protein, U6 snRNA, poly(A) and viral RNA, is the stretch of 'bulged-out' A residues. Thus, the NS1 protein was found to recognize either the stretch of 'bulged-out' A residues or dsRNA which is also a target of the NS1 protein. Furthermore, a basic peptide, NS1-2, derived from the helix-2 of the RNA binding site of NS1 protein was designed and its binding to the U6 snRNA was analysed by using a model RNA for U6 snRNA, U6-34. The NMR signals due to H8/H6 and H1' of U6-34 were assigned and their changes upon binding of NS1-2 were analysed. It was indicated that NS1-2 interacts with the residues in the bulge-out region of U6-34. These results suggest that NS1-2 recognizes the U6 snRNA in a similar manner to NS1 protein.