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A Mayeda

Publications and source records attributed to A Mayeda.

49 records · Page 3Linked to original sources
PubMed1992

Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2.

When messenger RNA precursors (pre-mRNAs) containing alternative 5' splice sites are spliced in vitro, the relative concentrations of the heterogeneous ribonucleoprotein (hnRNP) A1 and the essential splicing factor SF2 precisely determine which 5' splice site is selected. In general, an excess of hnRNP A1 favors distal 5' splice sites, whereas an excess of SF2 results in utilization of proximal 5' splice sites. The regulation of these antagonistic activities may play an important role in the tissue-specific and developmental control of gene expression by alternative splicing.

Adenovirus Early Proteins↗
PubMed1991

Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators.

SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner. This latter property suggests a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing. Human SF2 cDNAs have been isolated and overexpressed in bacteria. Recombinant SF2 is active in splicing and stimulates proximal 5' splice sites. SF2 has a C-terminal region rich in arginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70K polypeptide and the Drosophila alternative splicing regulators transformer, transformer-2, and suppressor-of-white-apricot. Like transformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.

Amino Acid Sequence↗
PubMed1990

Beta-globin transcripts carrying a single intron with three adjacent nucleotides of 5' exon are efficiently spliced in vitro irrespective of intron position or surrounding exon sequences.

To examine the role of exon sequences and intron position in the splicing of an mRNA precursor, we prepared series of sense or anti-sense transcripts of human beta-globin cDNA in which a cassette containing the beta-globin first intron was inserted into one of seven unusual positions. The intron cassette consisted of the intron alone (ml), the intron with three adjacent base pairs of the 5' exon (MI), or the intron with both 5' and 3' exon sequences. All these transcripts were examined in an in vitro splicing system with a HeLa cell nuclear extract. The sense transcripts carrying MI cassette were spliced efficiently and independently of the intron position, except when the 3' exon was too short. The anti-sense transcripts carrying MI cassette produced significantly less spliced products than did those of the sense transcripts. This was mostly because of the instability of the anti-sense transcripts, and the actual splicing efficiency was similar to that seen in the sense transcripts. Sense or anti-sense transcripts carrying ml cassette were spliced to various extents depending on the surrounding sequences. The results indicate that only three nucleotides of the 5' exon are required as specific exon sequences in the splicing of an mRNA precursor carrying a single intron, and that the intron position does not significantly affect the splicing efficiency in vitro.

Base Sequence↗
PubMed1990

Surveying cis-acting sequences of pre-mRNA by adding antisense 2'-O-methyl oligoribonucleotides to a splicing reaction.

We chemically synthesized antisense 12 mer 2'-O-methylribonucleotides and surveyed a scanning (signal-tracking) process as well as sequences within a beta-globin transcript acting in the splicing reaction in vitro. The pre-mRNA transcript contained the sequences of the first exon, first intron, and a major part of the second exon of the human beta-globin gene. We found that the antisense 2'-O-methylribonucleotides could anneal effectively to the target site in the pre-mRNA during the splicing reaction. A 2'-O-methylribonucleotide complementary to the donor (5') splice site completely inhibited authentic splicing and activated an upstream cryptic donor site. A 2'-O-methylribonucleotide complementary to the branch site inhibited normal branch formation and greatly reduced subsequent generation of the spliced product. Six other 2'-O-methylribonucleotides complementary to loci in the exons or the intronic region between the donor and branch sites had no significant effect on the splicing reaction. These observations suggest that an extensive scanning of the present pre-mRNA across the six regions tested is not essential for the splicing reaction. We propose that a short antisense 2'-O-methylribonucleotide provides a practical and convenient method to examine cis-acting sequences of RNA. The advantages of this method in comparison with site-directed mutagenesis or deletion are discussed.

Base Sequence↗
PubMed1988

Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

Amino Acid Sequence↗
PubMed1986

Three distinct activities possibly involved in mRNA splicing are found in a nuclear fraction lacking U1 and U2 RNA.

A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography, and the fractions were assayed for the binding activity for a small RNA transcript carrying a splice junction or branch point sequence. The binding activity for the RNA carrying a 5' splice junction was localized in the small nuclear ribonucleoprotein (snRNP) fraction together with binding activity for the RNA carrying a 3' splice junction or branch point sequence. However, stronger binding activities for the 3' splice junction RNA and for the branch point RNA were discovered in the flow-through fraction where no small nuclear RNA were detected. When the flow-through fraction was added to purified U1 ribonucleoprotein, the binding activity for the 5' splice junction RNA was markedly enhanced. We propose that the factors responsible for the three types of activities found in the flow-through fraction play a role in the splicing of mRNA precursor.

Adenosine Triphosphate↗
PubMed1984

U1 RNA-protein complex preferentially binds to both 5' and 3' splice junction sequences in RNA or single-stranded DNA.

We have investigated factors that recognize the splice junctions for mRNA by means of a rapid and sensitive filter binding assay using chemically synthesized single-stranded (ss) DNA (16-21 nucleotides) that includes a splice junction sequence or using RNA transcribed from the DNA. When small nuclear RNA-protein complexes from HeLa cells or rat liver were separated by a DEAE-Sepharose column, U1 RNA-protein complex fractions showed strong binding to ss DNA including a 5' or 3' consensus splice junction sequence. This binding took place in the presence of a large excess of Escherichia coli denatured DNA or RNA, but it was significantly reduced when conserved G-T or A-G within the splice junction was altered. In contrast, the U2 RNA-protein complex fractions did not show significant binding. We also have prepared RNA carrying the splice junction sequence by in vitro transcription of double-stranded splice junction DNA, which was linked to the E. coli lac promoter. By using this RNA, preferential binding to both 5' and 3' splice junction sequences has been confirmed with the partially purified U1 RNA-protein complex fraction described above. When the U1 RNA-protein complex is highly purified, it always retains a strong binding activity for a 5' splice junction. The binding activity for a 3' splice junction is partly or mostly lost during purification. These results strongly suggest that the U1 RNA-protein complex and/or an associated factor participates in the recognition of both 5' and 3' splice junctions.

Animals↗