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Phosphorylated adaptor for RNA export (PHAX) is the key export mediator for spliceosomal U small nuclear RNA (snRNA) precursors in metazoa. PHAX is enriched in Cajal bodies (CBs), nuclear subdomains involved in the biogenesis of small ribonucleoproteins. However, CBs’ role in U snRNA export has not been demonstrated. In this study, we show that U snRNA precursors microinjected into Xenopus laevis oocyte nuclei temporarily concentrate in CBs but gradually decrease as RNA export proceeds. Inhibition of PHAX activity by the coinjection of a specific anti-PHAX antibody or a dominant-negative PHAX mutant inhibits U snRNA export and simultaneously enhances accumulation of U snRNA precursors in CBs, indicating that U snRNAs transit through CBs before export and that binding to PHAX is required for efficient exit of U snRNAs from CBs. Similar results were obtained with U snRNAs transcribed from microinjected genes. These results reveal a novel function for CBs, which ensure that U snRNA precursors are properly bound by PHAX. 相似文献
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U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3′ end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3′ end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting. 相似文献
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Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs
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Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm. 相似文献
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Simoes-Barbosa A Meloni D Wohlschlegel JA Konarska MM Johnson PJ 《RNA (New York, N.Y.)》2008,14(8):1617-1631
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. 相似文献
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Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns''own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns. 相似文献
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The 5′-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2,7-trimethylguanosine structure. 5′-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5′-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C′ in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C′ in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C′ in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs. 相似文献
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Using an in vitro system we have recently shown that the 3′ ends of human pre-snRNAs synthesized by RNA polymerase II are produced by RNA processing directed by the snRNA gene-specific 3′ box. Towards a complete characterization of this processing reaction we have further investigated the in vitro requirements for proper 3′ end formation of pre-U1 snRNA. Here we show that the 5′ cap plays a stimulatory role and processing requires creatine phosphate. Our results also indicate that the pre-U1 processing activity is heat sensitive and that an RNA component is required. In addition, the exact sequence adjacent to the 3′ box influences the position of the pre-U1 3′ end produced in vitro. Interestingly, the processing extract active for 3′-box-dependent processing also contains an activity that converts the 3′ end of RNA containing the U1 Sm protein binding site and the 3′ terminal stem–loop into the mature form. 相似文献
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Kyriakopoulou C Larsson P Liu L Schuster J Söderbom F Kirsebom LA Virtanen A 《RNA (New York, N.Y.)》2006,12(9):1603-1611
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. 相似文献
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Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA. 相似文献
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Xueni Li Wenzheng Zhang Tao Xu Jolene Ramsey Lingdi Zhang Ryan Hill Kirk C. Hansen Jay R. Hesselberth Rui Zhao 《Nucleic acids research》2013,41(6):3805-3818
Prp8 stands out among hundreds of splicing factors as a protein that is intimately involved in spliceosomal activation and the catalytic reaction. Here, we present the first comprehensive in vivo RNA footprints for Prp8 in budding yeast obtained using CLIP (cross-linking and immunoprecipitation)/CRAC (cross-linking and analyses of cDNAs) and next-generation DNA sequencing. These footprints encompass known direct Prp8-binding sites on U5, U6 snRNA and intron-containing pre-mRNAs identified using site-directed cross-linking with in vitro assembled small nuclear ribonucleoproteins (snRNPs) or spliceosome. Furthermore, our results revealed novel Prp8-binding sites on U1 and U2 snRNAs. We demonstrate that Prp8 directly cross-links with U2, U5 and U6 snRNAs and pre-mRNA in purified activated spliceosomes, placing Prp8 in position to bring the components of the active site together. In addition, disruption of the Prp8 and U1 snRNA interaction reduces tri-snRNP level in the spliceosome, suggesting a previously unknown role of Prp8 in spliceosomal assembly through its interaction with U1 snRNA. 相似文献
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Although spliceosomal introns are an abundant landmark in eukaryotic genomes, the nuclear genome of the divergent eukaryote Giardia intestinalis, the causative agent of giardiasis, has been considered as “intron-poor” with only five canonical (cis-spliced) introns. However, three research groups (including ours) have independently reported a novel class of spliceosomal introns in the G. intestinalis genome. Three protein-coding genes are split into pieces in the G. intestinalis genome, and each of the partial coding regions was independently transcribed into polyadenylated premature mRNAs (pre-mRNAs). The two pre-mRNAs directly interact with each other by an intermolecular-stem structure formed between their non-coding portions, and are then processed into mature mRNAs by spliceosome-mediated trans-splicing. Here, we summarize the recently published works on split introns (“splintrons”) in the G. intestinalis genome, and then provide our speculation on the functional property of the Giardia spliceosomes based on the putative ratio of splintrons to canonical introns. Finally, we discuss a scenario for the transition from typical GT-AG boundaries to non-typical AT-AC boundaries in a particular splintron of Giardia. 相似文献
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Structural basis for the dual U4 and U4atac snRNA-binding specificity of spliceosomal protein hPrp31
Human proteins 15.5K and hPrp31 are components of the major spliceosomal U4 snRNP and of the minor spliceosomal U4atac snRNP. The two proteins bind to related 5'-stem loops (5'SLs) of the U4 and U4atac snRNAs in a strictly sequential fashion. The primary binding 15.5K protein binds at K-turns that exhibit identical sequences in the two snRNAs. However, RNA sequences contacted by the secondary binding hPrp31 differ in U4 and U4atac snRNAs, and the mechanism by which hPrp31 achieves its dual specificity is presently unknown. We show by crystal structure analysis that the capping pentaloops of the U4 and U4atac 5'SLs adopt different structures in the ternary hPrp31-15.5K-snRNA complexes. In U4atac snRNA, a noncanonical base pair forms across the pentaloop, based on which the RNA establishes more intimate interactions with hPrp31 compared with U4 snRNA. Stacking of hPrp31-His270 on the noncanonical base pair at the base of the U4atac pentaloop recapitulates intramolecular stabilizing principles known from the UUCG and GNRA families of RNA tetraloops. Rational mutagenesis corroborated the importance of the noncanonical base pair and the U4atac-specific hPrp31-RNA interactions for complex stability. The more extensive hPrp31-U4atac snRNA interactions are in line with a higher stability of the U4atac compared with the U4-based ternary complex seen in gel-shift assays, which may explain how U4atac snRNA can compete with the more abundant U4 snRNA for the same protein partners in vivo. 相似文献
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Jessica Tsalikis Ivan Tattoli Arthur Ling Matthew T. Sorbara David O. Croitoru Dana J. Philpott Stephen E. Girardin 《The Journal of biological chemistry》2015,290(34):20904-20918
Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense. 相似文献
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Simoes-Barbosa A Louly C Franco OL Rubio MA Alfonzo JD Johnson PJ 《Nucleic acids research》2008,36(21):6848-6858
Eukaryotic RNAs typically contain 5′ cap structures that have been primarily studied in yeast and metazoa. The only known RNA cap structure in unicellular protists is the unusual Cap4 on Trypanosoma brucei mRNAs. We have found that T. vaginalis mRNAs are protected by a 5′ cap structure, however, contrary to that typical for eukaryotes, T. vaginalis spliceosomal snRNAs lack a cap and may contain 5′ monophophates. The distinctive 2,2,7-trimethylguanosine (TMG) cap structure usually found on snRNAs and snoRNAs is produced by hypermethylation of an m7G cap catalyzed by the enzyme trimethylguanosine synthase (Tgs). Here, we biochemically characterize the single T. vaginalis Tgs (TvTgs) encoded in its genome and demonstrate that TvTgs exhibits substrate specificity and amino acid requirements typical of an RNA cap-specific, m7G-dependent N2 methyltransferase. However, recombinant TvTgs is capable of catalysing only a single round of N2 methylation forming a 2,7-dimethylguanosine cap (DMG) as observed previously for Giardia lamblia. In contrast, recombinant Entamoeba histolytica and Trypanosoma brucei Tgs are capable of catalysing the formation of a TMG cap. These data suggest the presence of RNAs with a distinctive 5′ DMG cap in Trichomonas and Giardia lineages that are absent in other protist lineages. 相似文献