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At least 793 records · Page 44Linked to original sources

PubMed2001

Non-coding snoRNA host genes in Drosophila: expression strategies for modification guide snoRNAs.

Modification guide snoRNAs either are encoded within introns and co-transcribed with the host gene pre-mRNA or are independently transcribed as mono- or polycistronic units. Different eukaryotic kingdoms utilize these coding strategies to various degrees. Intron-encoded and polycistronic snoRNAs are released from primary transcripts as pre-snoRNAs by the spliceosome or by an RNase III-like activity, respectively. In the spliceosomal pathway, the resulting intron lariat is then linearized by a debranching activity. The leader and trailer sequences of pre-snoRNAs are removed by exonucleolytic activities. The majority of snoRNA host genes encode proteins involved in the synthesis, structure or function of the translational apparatus. Several vertebrate snoRNA host genes do not appear to code for functional proteins. We have identified two unusually compact box C/D multi-snoRNA host genes in D. melanogaster, dUHG1 and dUHG2, similar in their organization to the corresponding vertebrate non-protein-coding host genes. In dUHG1 and dUHG2, the snoRNA sequences are located within introns at a conserved distance of about 75 nucleotides upstream of the 3' splice sites. Both genes initiate transcription with TOP-like sequences that share unique features with previously reported Drosophila snoRNA host genes. Although the spliced dUHG RNAs are relatively stable, they exhibit little potential for protein coding.

Animals↗

PubMed2002

The structure and dynamics of the U4/U6 snRNP: implications for pre-mRNA splicing and use as a model system to investigate the RNA-mediated effects of (5F)Ura.

Pre-mRNA splicing is one of the most complex and intricate processes in eukaryotic cell biology. Over the past decade, my laboratory has been interested in determining the structures of RNA components of the spliceosome, and in investigating how the structure, stability and dynamics of these RNA components are perturbed by nucleoside analog substitution. In particular, we have investigated the U4/U6 snRNA complex as a model system for understanding the biophysical basis for the RNA-mediated effects of the widely-used anticancer drug 5-fluorouracil ((5F)Ura). In this review, our studies that have provided novel information concerning the structure of U4 snRNA and its interactions with U6 snRNA and the Sm (or common) snRNA binding proteins are summarized. These studies have also quantified the structural and thermodynamic consequences of (5F)Ura in this model system. Our work to date provides the foundation on which future studies investigating the biophysical basis for spliceosomal assembly and for clarifying the mechanisms of anticancer drugs targeted at nucleic acid-mediated processes will be developed.

Base Sequence↗

PubMed1997

Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

Animals↗

PubMed1997

In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein.

Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.

Base Composition↗

PubMed1999

Characterization of Sm-like proteins in yeast and their association with U6 snRNA.

Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis. Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA. Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses. Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA. Analyses of U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNP in the association-dissociation cycle of spliceosome complexes. Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function. We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing.

Amino Acid Sequence↗

PubMed2001

Proximity of the invariant loop of U5 snRNA to the second intron residue during pre-mRNA splicing.

A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.

Animals↗

PubMed2002

A cyclophilin functions in pre-mRNA splicing.

We report that the cyclophilin USA-CyP is part of distinct complexes with two spliceosomal proteins and is involved in both steps of pre-mRNA splicing. The splicing factors hPrp18 and hPrp4 have a short region of homology that defines a high affinity binding site for USA-CyP in each protein. USA-CyP forms separate, stable complexes with hPrp18 and hPrp4 in which the active site of the cyclophilin is exposed. The cyclophilin inhibitor cyclosporin A slows pre-mRNA splicing in vitro, and we show that its inhibition of the second step of splicing is caused by blocking the action of USA-CyP within its complex with hPrp18. Cyclosporin A also slows splicing in vivo, and we show that this slowing results specifically from inhibition of USA-CyP. Our results lead to a model in which USA-CyP is carried into the spliceosome in complexes with hPrp4 and hPrp18, and USA-CyP acts during splicing within these complexes. These results provide an example of the function of a cyclophilin in a complex process and provide insight into the mechanisms of action of cyclophilins.

Amino Acid Sequence↗

PubMed2002

Sequence-specific interaction of U1 snRNA with the SMN complex.

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.

Animals↗

PubMed2002

The splicing of U12-type introns can be a rate-limiting step in gene expression.

Some protein-coding genes in metazoan genomes contain a minor class of introns that are excised by a distinct, low-abundance spliceosome. We have developed a quantitative RT-PCR assay that allows comparison of the relative rates of intron removal from the transcripts present in a pre-mRNA population. We show that the U12-type introns are more slowly spliced than the major-class (U2-type) introns from three endogenous pre-mRNAs in human tissue culture cells. In Drosophila melanogaster S2 cells, using minigene constructs designed to produce nearly identical mRNAs, we observe increased expression of fluorescent protein and mature mRNA upon mutation of a U12-type to a U2-type intron. These results provide evidence that the level of gene expression in vivo is lowered by the presence of a U12-type intron and implicate the U12-type spliceosome as a target in the post-transcriptional regulation of gene expression.

Animals↗

PubMed1996

Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3' splice site selection.

PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the uridine tract that precedes most yeast 3' slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3' splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3' splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the prp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3' splice site selection and fidelity of 3' splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.

Amino Acid Sequence↗

PubMed1998

The domain encoded by exon 2 of the survival motor neuron protein mediates nucleic acid binding.

Spinal muscular atrophy (SMA) is a motor neuron disorder resulting from anterior horn cell death. Survival motor neuron ( SMN ) is the SMA-determining gene and is deleted or gene converted in >95% of SMA patients. The SMN protein has a role in spliceosomal snRNP biogenesis and has therefore been implicated indirectly in general cellular RNA processing due to its unique sub-nuclear localization within structures termed 'gems', which co-localize with spliceosomal factors within coiled bodies. In this report, direct SMN RNA-binding activity, in addition to ssDNA and dsDNA binding is demonstrated. The region of SMN encoded by exon 2 is necessary and sufficient to mediate its nucleic acid-binding activities. This domain is homologous to several nucleic acid-binding factors, including several high mobility group (HMG) proteins. Additionally, previously reported SMN missense mutations isolated from SMA patients demonstrated reduced RNA-binding activity, suggesting that nucleic acid binding is functionally significant.

Amino Acid Sequence↗

PubMed1990

Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes.

By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5' end) and direct RNA sequencing of purified U3 RNA from tomato (3' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.

Base Sequence↗

PubMed1991

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.

Base Sequence↗

PubMed1992

Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing.

Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop Ila region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.

Base Sequence↗

PubMed1995

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.

Animals↗

PubMed1998

A 7-methylguanosine cap commits U3 and U8 small nuclear RNAs to the nucleolar localization pathway.

U3 and U8 small nucleolar RNAs (snRNAs) participate in pre-rRNA processing. Like the U1, U2, U4 and U5 major spliceosomal snRNAs, U3 and U8 RNAs are transcribed by RNA polymerase II and their initial 7-methylguanosine (m7G) 5' cap structures subsequently become converted to 2,2,7-trimethylguanosine. However, unlike the polymerase II transcribed spliceosomal snRNAs, which are exported to the cytoplasm for cap hypermethylation, U3 and U8 RNAs undergo cap hypermethylation within the nucleus. Human U3 and U8 RNAs with various cap structures were generated by in vitro transcription, fluorescently labeled and microinjected into nuclei of normal rat kidney (NRK) epithelial cells. When U3 and U8 RNAs containing a m7G cap were microinjected they became extensively localized in nucleoli. U3 and U8 RNAs containing alternative cap structures did not localize in nucleoli nor did U3 or U8 RNAs containing triphosphate 5'-termini. The nucleolar localization of m7G-capped U3 RNA was competed by co-microinjection into the nucleus of a 100-fold molar excess of dinucleotide m7GpppG but not by a 100-fold excess of ApppG dinucleotide. Although it was obviously not possible to assess formation of di- and trimethylguanosine caps on the microinjected U3 and U8 RNAs in these single cell experiments, these results indicate that the initial presence of a m7G cap on U3 and U8 RNAs, most likely together with internal sequence elements, commits these transcripts to the nucleolar localization pathway and point to diverse roles of the m7G cap in the intracellular traffic of various RNAs transcribed by RNA polymerase II.

Animals↗

PubMed1999

Concerted regulation and molecular evolution of the duplicated SNRPB'/B and SNRPN loci.

The human small nuclear ribonucleoprotein SNRPB ' /B gene is alternatively spliced to produce the SmB or SmB' spliceosomal core proteins. An ancestral duplication gave rise to the closely related SNRPN paralog whose protein product, SmN, replaces SmB'/B in brain. However, the precise evolutionary and functional relationship between these loci has not been clear. Genomic, cDNA and protein analyses presented here in chicken, two marsupials (South American opossum and tammar wallaby), and hedgehog, suggest that the vertebrate ancestral locus produced the SmB' isoform. Interestingly, three eutherians exhibit radically distinct splice choice expression profiles, producing either exclusively SmB in mouse, both SmB and SmB' in human, or exclusively SmB' in hedgehog. The human SNRPB ' /B locus is biallelically unmethylated, unlike the imprinted SNRPN locus which is unmethyl-ated only on the expressed paternal allele. Western analysis demonstrates that a compensatory feedback loop dramatically upregulates SmB'/B levels in response to the loss of SmN in Prader-Willi syndrome brain tissue, potentially reducing the phenotypic severity of this syndrome. These findings imply that these two genes encoding small nuclear ribonucleoprotein components are subject to dosage compensation. Therefore, a more global regulatory network may govern the maintenance of stoichiometric levels of spliceosomal components and may constrain their evolution.

Alternative Splicing↗

PubMed2001

A computational scan for U12-dependent introns in the human genome sequence.

U12-dependent introns are found in small numbers in most eukaryotic genomes, but their scarcity makes accurate characterisation of their properties challenging. A computational search for U12-dependent introns was performed using the draft version of the human genome sequence. Human expressed sequences confirmed 404 U12-dependent introns within the human genome, a 6-fold increase over the total number of non-redundant U12-dependent introns previously identified in all genomes. Although most of these introns had AT-AC or GT-AG terminal dinucleotides, small numbers of introns with a surprising diversity of termini were found, suggesting that many of the non-canonical introns found in the human genome may be variants of U12-dependent introns and, thus, spliced by the minor spliceosome. Comparisons with U2-dependent introns revealed that the U12-dependent intron set lacks the 'short intron' peak characteristic of U2-dependent introns. Analysis of this U12-dependent intron set confirmed reports of a biased distribution of U12-dependent introns in the genome and allowed the identification of several alternative splicing events as well as a surprising number of apparent splicing errors. This new larger reference set of U12-dependent introns will serve as a resource for future studies of both the properties and evolution of the U12 spliceosome.

Alternative Splicing↗