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At least 1,153 records · Page 64Linked to original sources
PubMed1994

Protein phosphatase 1 can modulate alternative 5' splice site selection in a HeLa splicing extract.

Recent studies using HeLa in vitro splicing extracts have shown that changes in the relative concentrations of constitutive protein splicing factors can affect the choice between competing 5' splice sites in alternatively spliced mammalian pre-mRNAs. Here we report that treatment of a HeLa splicing extract with human protein phosphatase 1 strongly inhibits formation of mRNA spliced to the distal 5' splice site while stimulating relative use of the proximal 5' splice site. This effect is not observed if spliceosomes assemble prior to protein phosphatase 1 treatment. These data show that alternative splicing in HeLa extracts can be mediated by changes in protein modification as well as by changes in the relative concentration of splicing factors. Changes in protein phosphorylation may thus provide a rapid mechanism for cells to respond to stimuli that require an alteration in alternative splicing patterns.

Alternative Splicing↗
PubMed1988

Alternative use of a polyadenylation signal and of a downstream 3' splice site. Effect of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site P1 instead of the major promotor-distal sites P2 and P4. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of P1. The nascent transcript is first cleaved in the branch point-3' splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3' end (F-RNA) and a promoter-distal RNA with a heterogeneous 5' end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3' end of F-RNA is about 130 nucleotides downstream from P1 and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CP1, which is the transcript ending at P1. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3' end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.

Adenoviridae↗
PubMed1991

Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA.

The yeast mitochondrial group II intron bI1 is self-splicing in vitro. We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons. Since exon reopening is supposed to correspond to a reverse 3' cleavage this suggests that the deletion specifically blocks the first reaction step. The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron. These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro. Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.

Base Sequence↗
PubMed1992

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.

Autoantigens↗
PubMed1986

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro.

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.

Base Sequence↗
PubMed1987

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.

Animal Population Groups↗
PubMed1987

S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA.

We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.

Animal Population Groups↗
PubMed1989

Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides.

We have used antisense 2'-OMe RNA oligonucleotides carrying four 5'-terminal biotin residues to probe the structure and function of the human U4/U6 snRNP. Nine oligonucleotides, complementary to multiple regions of U4 and U6 snRNAs, bound stably and specifically to U4/U6 snRNP. This allowed for efficient and selective removal of U4/U6 from HeLa cell nuclear extracts. Binding of oligonucleotides to certain snRNA domains inhibited splicing and affected the U4-U6 interaction. Pre-mRNA and splicing products could also be affinity-selected through binding of the oligonucleotides to U4/U6 snRNPs in splicing complexes. The results suggest that U4 snRNP is not released during spliceosome assembly.

Base Sequence↗
PubMed1989

Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing.

Alternative splicing of alpha-tropomyosin pre-mRNA involves mutually exclusive utilization of exons 2 and 3, exon 3 being preferentially selected in most cells. This mutually exclusive behavior is enforced by absolute incompatibility between the adjacent splice sites of the two exons, due to close proximity of the exon 3 branch point to exon 2. The branch point, with an associated polypyrimidine tract, is in an unusual location, 177 nt upstream of the acceptor, only 42 nt from the exon 2 splice donor site. Splicing of exon 2 to 3 is consequently blocked prior to formation of an active spliceosome complex. This block to splicing can be relieved by insertion of spacer elements that increase the donor site-branch point separation to 51-59 nt. The unconventional relative location of the constitutive cis splicing elements therefore provides a simple mechanistic basis for strict mutually exclusive splicing. These results not only demonstrate that the branch point is not specified by proximity to the splice acceptor site, but rather suggest that it is the acceptor site which is specified relative to the branch point.

Animals↗
PubMed1990

A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing.

We previously described a dominant suppressor of the splicing defect conferred by an A----C intron branchpoint mutation in S. cerevisiae. Suppression occurs by increasing the frequency with which the mutant branchpoint is utilized. We have now cloned the genomic region encoding the prp16-1 suppressor function and have demonstrated that PRP16 is essential for viability. A 1071 amino acid open reading frame contains sequence motifs characteristic of an NTP binding fold and further similarities to a superfamily of proteins that includes members with demonstrated RNA-dependent ATPase activity. A single nucleotide change necessary to confer the prp16-1 suppressor phenotype results in a Tyr----Asp substitution near the "A site" consensus for NTP binding proteins. We propose that PRP16 is an excellent candidate for mediating one of the many ATP-requiring steps of spliceosome assembly and that accuracy of branchpoint recognition may be coupled to ATP binding and/or hydrolysis.

Adenosine Triphosphatases↗
PubMed1990

Spliced leader RNA sequences can substitute for the essential 5' end of U1 RNA during splicing in a mammalian in vitro system.

L. collosoma or C. elegans SL RNA sequences joined to an adenovirus intron and 3' exon are spliced highly efficiently and accurately in HeLa nuclear extract. After inactivation of U1 snRNPs using RNAase H and a deoxyoligonucleotide complementary to the first 12 nucleotides of U1, splicing of SL RNA-containing constructs continues undiminished, whereas control substrates no longer splice. Since neither binding of U1 snRNPs nor inhibition of splicing is detected using anti-(U1)RNP antibodies, splicing of SL RNA-containing constructs may be entirely U1 snRNP independent. Analyses of altered L. collosoma constructs revealed that the sequence surrounding the 5' splice site is not sufficient to confer U1-independent splicing; the smallest U1-independent region identified so far retains only the first stem-loop of the SL RNA. That sequences responsible for recognition of the 5' splice site can be relocated within the splicing substrate itself reinforces the similarity between group II self-splicing and spliceosome-mediated pre-mRNA splicing.

Animals↗
PubMed1991

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev.

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.

Amino Acid Sequence↗
PubMed1994

Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription.

The 40 kb Balbiani ring 1 (BR1) gene is at a given moment transcribed by, on average, 120 RNA polymerases. Here we directly assay the excision of introns both in the nascent and in the released nucleoplasmic BR1 pre-mRNAs, isolated by microdissection. We show that intron 3, located 3 kb from the 5' end of the pre-mRNA, is excised simultaneous with transcription. Within 2.5 min of transcription time, 50% of the pre-mRNA molecules have lost the intron. Intron 4, located 600 bases from the polyadenylation site, is excised cotranscriptionally in 5%-10% of the molecules and after or during release to the nucleoplasm in the remaining molecules. Our results demonstrate that spliceosome assembly is a cotranscriptional process in vivo and that splicing may occur during transcription but also after completed transcription, depending on the position of the intron.

Animals↗
PubMed1995

Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain.

The yeast mitochondrial group I intron b15 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions. Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure. Folding energy provided by CBP2 drives assembly of two RNA domains that comprise the catalytic core and meditates association of an approximately 100 nt 5' domain that contains the 5' splice site. Thus, the protein assembles RNA secondary structure elements into a specific three-dimensional array while the RNA provides the catalytic center. The division of labor between RNA and protein illustrated by this simple system reveals principles applicable to complex ribonucleoprotein assemblies such as the spliceosome and ribosome.

Base Sequence↗
PubMed1995

The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs.

HIV-1 Rev protein directs nuclear export of pre-mRNAs and mRNAs containing its binding site, the Rev response element (RRE). To define how Rev acts, we used conjugates between bovine serum albumin (BSA) and peptides comprising the Rev activation domain (BSA-R). BSA-R inhibited Rev-mediated nuclear RNA export, whereas a mutant activation domain peptide conjugate did not. BSA-R did not affect the export of mRNA, tRNA, or ribosomal subunits, but did inhibit export of 5S rRNA and spliceosomal U snRNAs. BSA-R was itself exported from the nucleus in an active, saturable manner. Thus, the Rev activation domain constitutes a nuclear export signal that redirects RRE-containing viral RNAs to a non-mRNA export pathway.

Amino Acid Sequence↗
PubMed1994

Defective splicing induced by 4NQO in the hamster hprt gene.

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.

4-Nitroquinoline-1-oxide↗
PubMedDate not supplied

Characterisation of genes encoding a nucleoside monophosphate kinase and a L35 ribosomal protein from Babesia bovis.

We have sequenced a region of the Babesia bovis nuclear genome that encodes a L35 ribosomal protein homologue (bl35) and a putative nucleoside monophosphate kinase (bnmk) that is most similar to the adenylate kinase of gram-positive bacteria and the mitochondrial form of adenylate kinase in eukaryotes. BNMK appears to be unique in that it is the first eukaryotic family member to feature a putative zinc-binding domain. bnmk and bl35 are closely linked and transcribed from opposite DNA strands. Examination of the gene structures indicate that the coding regions contain small intervening sequences that obey the GT-AG rule of eukaryotic spliceosomal introns. The single intron separates the bl35 initiation codon from the remainder of the coding region and the 6-exon bnmk gene does not appear to be differentially spliced. Both genes utilise multiple polyadenylation sites and the canonical mammalian polyadenylation signal AATAAA is absent from their 3' untranslated regions. Primer extension analyses reveal that the bnmk gene utilises a cluster of transcription start points, one of which is used most frequently. The bnmk mRNA 5' end does not appear to be cis- or trans-spliced. We report here the first evidence of intronic sequences, as well as heterogeneous 5' and 3' ends for mRNA of a member of the Babesia genus.

Amino Acid Sequence↗
PubMed1994

Pseudouridine formation in small nuclear RNAs.

Recent in vitro studies on the formation of pseudouridine (psi) in the spliceosomal small nuclear RNAs (snRNA) are reviewed. Multiple psi synthase activities, in some cases more that one per snRNA, are responsible for this modification of uridine. There is a requirement for Sm protein binding for the efficient formation of psi in U5 RNA but not for the modification of U2 RNA. The inhibition of psi formation by the incorporation of 5-fluorouridine in the snRNA is also reviewed.

Animals↗