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.     

Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region

The U2 small nuclear RNA (snRNA) of Trypanosoma brucei gambiense, a flagellated protozoon of the order Kinetoplastida, is 148 nucleotides (nt) long, and thus the smallest U2 snRNA identified so far. To examine the evolutionary conservation of this RNA among Kinetoplastida, we have cloned and sequenced the U2 genes from Trypanosoma congolense and Leishmania mexicana amazonensis, which are 145 and 141 nt in length, respectively. The sequences of the Kinetoplastida U2 snRNAs are essentially identical in the 5' half of the molecule. Surprisingly, the putative branch site recognition sequence of L. m. amazonensis U2 snRNA shows two nt changes when compared with the other two U2 snRNAs. The sequence of the 3' half of the Kinetoplastida U2 snRNAs is less conserved with T. congolense and L. m. amazonensis RNAs showing 23 and 35 nt sequence variations, respectively, when compared with the corresponding sequence of the T. b. gambiense U2 snRNA. Alignment of the flanking regions of the U2 genes revealed several elements which are conserved both in sequence and in position relative to the U2 coding region and which may function in the biosynthesis of U2 snRNAs. One upstream element specifically binds protein factor(s) present in T. brucei nuclear extracts.     

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.     

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.     

Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes.

We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila. The genes of the individual snRNAs exist in two to six slightly different copies per haploid genome. Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes. Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes. Comparison of the promoter structure of the snRNA genes of T. thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms. This finding is discussed in relation to a possible role in snRNA 3'-end formation.     

Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of 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.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

cDNA cloning of U1, U2, U4 and U5 snRNA families expressed in pea nuclei.

Differences observed between plant and animal pre-mRNA splicing may be the result of primary or secondary structure differences in small nuclear RNAs (snRNAs). A cDNA library of pea snRNAs was constructed from anti-trimethylguanosine (m3(2,2,7)G immunoprecipitated pea nuclear RNA. The cDNA library was screened using oligo-deoxyribonucleotide probes specific for the U1, U2, U4 and U5 snRNAs. cDNA clones representing U1, U2, U4 and U5 snRNAs expressed in seedling tissue have been isolated and sequenced. Comparison of the pea snRNA variants with other organisms suggest that functionally important primary sequences are conserved phylogenetically even though the overall sequences have diverged substantially. Structural variations in U1 snRNA occur in regions required for U1-specific protein binding. In light of this sequence analysis, it is clear that the dicot snRNA variants do not differ in sequences implicated in RNA:RNA interactions with pre-mRNA. Instead, sequence differences occur in regions implicated in the binding of small ribonucleoproteins (snRNPs) to snRNAs and may result in the formation of unique snRNP particles.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.     

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.     

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.     

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5'' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5'' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5'' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.     

植物核小RNA研究进展

核小RNA (small nuclear RNA,snRA)是一类长度为60～300 nt的非编码RNA,是真核生物RNA剪接体的主要成分. snRNA在各种生物中具有较高的保守性和同源性,主要参与了mRNA和rRNA前体的加工过程.本文对当前植物中snRNA领域的研究成果进行简要概述,介绍了植物snRNA的分类、基因结构、合成机制及生物学功能,同时总结了植物snRNA的化学修饰以及在科研生产中的应用,并对将来snRNA的研究方向进行了展望.     

Maize U2 snRNAs: gene sequence and expression.

The complexity of plant U-type small nuclear ribonucleoprotein particles (UsnRNPs) may represent one level at which differences in splicing between animals and plants and between monocotyledonous and dicotyledonous plants could be effected. The maize (monocot.) U2snRNA multigene family consists of some 25 to 40 genes which from RNA blot and RNase protection analyses produce U2snRNAs varying in both size and sequence. The first 77 nucleotides of the maize U2-27 snRNA gene are identical to U2snRNA genes of Arabidopsis (dicot). Despite much lower sequence homology in the remaining 120 nucleotides the secondary structure of the RNA is conserved. The difference in splicing between monocot. and dicot. plants cannot be explained on the basis of sequence differences between monocot, and dicot. U2snRNAs in the region which may interact with intron branch point sequences.