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.     

A common maturation pathway for small nucleolar RNAs.

We have shown that precursors of U3, U8 and U14 small nucleolar RNAs (snoRNAs) are not exported to the cytoplasm after injection into Xenopus oocyte nuclei but are selectively retained and matured in the nucleus, where they function in pre-rRNA processing. Our results demonstrate that Box D, a conserved sequence element found in these and most other snoRNAs, plays a key role in their nuclear retention, 5' cap hypermethylation and stability. Retention of U3 and U8 RNAs in the nucleus is saturable and relies on one or more common factors. Hypermethylation of the 5' caps of U3 RNA occurs efficiently in oocyte nuclear extracts lacking nucleoli, suggesting that precursor snoRNAs are matured in the nucleoplasm before they are localized to the nucleolus. Surprisingly, m7G-capped precursors of spliceosomal small nuclear RNAs (snRNAs) such as pre-U1 and U2, can be hypermethylated in nuclei if the RNAs are complexed with Sm proteins. This raises the possibility that a single nuclear hypermethylase activity may act on both nucleolar and spliceosomal snRNPs.     

A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import.

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import. Previous studies have demonstrated that the assembly of pre-snRNAs into ribonucleoprotein particles containing the Sm core proteins is essential for nuclear import in mammalian cells but that 5' cap hypermethylation is not. In the present investigation we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' tails, and identified one that was stalled (or greatly slowed) in 3' end processing, leading to its accumulation in the cytoplasm of human cells. Nonetheless, this 3' processing arrested pre-U2 RNA molecule was found to undergo cytoplasmic assembly into Sm protein-containing complexes to the same extent as normal pre-U2 RNA. The Sm protein-associated, unprocessed mutant pre-U2 RNA was not observed in the nuclear fraction. Using an assay based on suppression of a genetically blocked SV40 pre-mRNA splicing pathway, we found that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.     

U1-like snRNAs lacking complementarity to canonical 5' splice sites

We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.     

Cross-linking of U2 snRNA using nitrogen mustard. Evidence for higher order structure.

Nuclear mRNA precursors are spliced by a large macromolecular complex called the spliceosome which contains, in most eucaryotes, five small nuclear RNAs (snRNAs) each in the form of a small ribonucleoprotein particle (the U1, U2, U5, and U4/U6 snRNPs). Although secondary structures have been derived for all five spliceosomal snRNAs based on phylogenetic, biochemical, and genetic data, little tertiary structure information is available. Here we use the general cross-linking reagent nitrogen mustard [bis-(2-chloroethyl)methylamine] to detect tertiary interactions within U2 snRNA. After the cross-linking of deproteinized HeLa nuclear extract, two intramolecularly cross-linked U2 species with anomalous electrophoretic mobility can be detected (X-U2#1 and X-U2#2). The 3' and 5' boundaries of each cross-link were determined by rapid enzymatic RNA sequencing of end-labeled RNA. X-U2#1 is cross-linked between the region U41-U55 and G105 or G106, X-U2#2 between U53 and G97 or G98. We then tested the ability of the two cross-linked species to bind snRNP proteins in vitro (in nuclear extract or S100) and in vivo (in Xenopus oocytes). X-U2#2 reconstituted efficiently both in vitro and in vivo but X-U2#1 did not, as judged by immunoprecipitation with antibodies specific for Sm- and U2-specific proteins. Since the cross-link in X-U2#2 involves the Sm binding site but does not block snRNP assembly, our data strongly suggest that the Sm binding site lies on the surface of the native snRNP.     

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei

In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.     

Nucleocytoplasmic transport and processing of small nuclear RNA precursors.

We have analyzed the structures and locations of small nuclear RNA (snRNA) precursors at various stages in their synthesis and maturation. In the nuclei of pulse-labeled Xenopus laevis oocytes, we detected snRNAs that were longer than their mature forms at their 3' ends by up to 10 nucleotides. Analysis of the 5' caps of these RNAs and pulse-chase experiments showed that these nuclear snRNAs were precursors of the cytoplasmic pre-snRNAs that have been observed in the past. Synthesis of pre-snRNAs was not abolished by wheat germ agglutinin, which inhibits export of the pre-snRNAs from the nucleus, indicating that synthesis of these RNAs is not obligatorily coupled to their export. Newly synthesized U1 RNAs could be exported from the nucleus regardless of the length of the 3' extension, but pre-U1 RNAs that were elongated at their 3' ends by more than about 10 nucleotides were poor substrates for trimming in the cytoplasm. The structure at the 3' end was critical for subsequent transport of the RNA back to the nucleus. This requirement ensures that truncated and incompletely processed U1 RNAs are excluded from the nucleus.     

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

Identification of 13 novel human modification guide RNAs

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.     

Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Characterization of the catalytic activity of U2 and U6 snRNAs

Removal of introns from pre-messenger RNAs in eukaryotes is carried out by the spliceosome, an assembly of a large number of proteins and five small nuclear RNAs (snRNAs). We showed previously that an in vitro transcribed and assembled base-paired complex of U2 and U6 snRNA segments catalyzes a reaction that resembles the first step of splicing. Upon incubation with a short RNA oligonucleotide containing the consensus sequence of the pre-mRNA branch site, the U2/U6 complex catalyzed a reaction between the 2' OH of a bulged adenosine and a phosphate in the catalytically important AGC triad of U6, leading to the formation of an X-shaped product, RNA X, apparently linked by an unusual phosphotriester bond. Here we characterize this splicing-related reaction further, showing that RNA X formation is an equilibrium reaction, and that the low yield of the reaction likely reflects an unfavorable equilibrium coefficient. Consistent with a phosphotriester linkage, RNA X is highly alkali-sensitive, but only mildly acid-sensitive. We also show that mutations in the AGC sequence of U6 can have significant effects on RNA X formation, further extending the similarities between splicing and RNA X formation. We also demonstrate that pseudouridylation of U2 enhances RNA X formation, and that U6 snRNA purified from nuclear extracts is capable of forming RNA X. Our data suggest that the ability to form RNA X might be an intrinsic property of spliceosomal snRNAs.