U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.

U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.     

A UV-crosslinkable interaction in human U6 snRNA.

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25-99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5' deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3' of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.     

Novel structure of a human U6 snRNA pseudogene

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.     

Control of mouse U1a and U1b snRNA gene expression by differential transcription.

The expression of mouse embryonic U1 snRNA (mU1b) genes is subject to stage- and tissue-specific control, being restricted to early embryos and adult tissues that contain a high proportion of stem cells capable of further differentiation. To determine the mechanism of this control we have sought to distinguish between differential RNA stability and regulation of U1 gene promoter activity in several cell types. We demonstrate here that mU1b RNA can accumulate to high levels in permanently transfected mouse 3T3 and C127 fibroblast cells which normally do not express the endogenous U1b genes, and apparently can do so without significantly interfering with cell growth. Expression of transfected chimeric U1 genes in such cells is much more efficient when their promoters are derived from a constitutively expressed mU1a gene rather than from an mU1b gene. In transgenic mice, introduced U1 transgenes with an mU1b 5' flanking region are subject to normal tissue-specific control, indicating that U1b promoter activity is restricted to tissues that normally express U1b genes. Inactivation of the embryonic genes during normal differentiation is not associated with methylation of upstream CpG-rich sequences; however, in NIH 3T3 fibroblasts, the 5' flanking regions of endogenous mU1b genes are completely methylated, indicating that DNA methylation serves to imprint the inactive state of the mU1b genes in cultured cells. Based on these results, we propose that the developmental control of U1b gene expression is due to differential activity of mU1a and mU1b promoters rather than to differential stability of U1a and U1b RNAs.     

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.     

利用人U6 snRNA启动子构建RNA干扰质粒载体

RNA干扰技术已经成为基因功能研究等领域的有力工具,构建带有筛选标记的siRNA载体可以在细胞中持续抑制靶基因的表达.为了利用RNAi技术开展生物学研究,在克隆载体pUC19的基础上改造构建了人类细胞小干扰RNA（small interference RNA,siRNA）表达质粒pUC19NU.该质粒具有新霉素抗性标记和真核细胞复制起点,利用连入的人U6 snRNA启动子起始siRNA的转录.以EGFP 和p53为靶基因的干扰实验证明,所构建的siRNA表达质粒可以显著抑制细胞外源性增强绿色荧光蛋白（enhanced green fluorescent protein,EGFP）及细胞内源性p53蛋白的表达,而且抑制效果具有特异性.     

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.     

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.     

An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA

Three yeast snRNAs (snR20, snR7, and snR14) have been implicated in pre-mRNA splicing. snR20 and snR7 contain domains of homology to U2 and U5, respectively, and each is required for viability. These RNAs are found associated with the spliceosome, as is snR14. We show here that snR14 is also an essential gene product. Sequence analysis reveals that, like snR7 and snR20, snR14 contains a consensus binding site for the Sm antigen, a feature common to all mammalian snRNAs involved in splicing. Moreover, snR14 exhibits several blocks of sequence and structural homology to U4, which in metazoans is found in association with U6. Native gel electrophoresis demonstrates that snR14 is in fact base-paired with another yeast snRNA, designated snR6, which has primary sequence homology to U6. We conclude that snR14 is the yeast analog of U4.     

Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.

A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes. Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.     

Structure of the yeast U2/U6 snRNA complex

The U2/U6 snRNA complex is a conserved and essential component of the active spliceosome that interacts with the pre-mRNA substrate and essential protein splicing factors to promote splicing catalysis. Here we have elucidated the solution structure of a 111-nucleotide U2/U6 complex using an approach that integrates SAXS, NMR, and molecular modeling. The U2/U6 structure contains a three-helix junction that forms an extended "Y" shape. The U6 internal stem-loop (ISL) forms a continuous stack with U2/U6 Helices Ib, Ia, and III. The coaxial stacking of Helix Ib on the U6 ISL is a configuration that is similar to the Domain V structure in group II introns. Interestingly, essential features of the complex--including the U80 metal binding site, AGC triad, and pre-mRNA recognition sites--localize to one face of the molecule. This observation suggests that the U2/U6 structure is well-suited for orienting substrate and cofactors during splicing catalysis.     

U2 and U6 snRNA genes in the microsporidian Nosema locustae: evidence for a functional spliceosome.

The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs). Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing. We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed. Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs. In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems. These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.