Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 7. Transfection of human YB-1 increased the inclusion of CD44 exons v4 and v5. (A) The reporter mini-gene containing CD44 variable exons v4 and v5. CD44 sequences are in black, human β-globin sequences are in gray, and the CMV promoter is in white. The CD44 insert is a contiguous genomic fragment mapping from 792 nucleotides upstream of exon v4 to 515 nucleotides downstream of exon v5. (B) Diagram of the wild-type and mutant YB-1 proteins made from co-expressing plasmids. The three indicated domains include an N-terminal alanine- and proline-rich region (AP), the single-stranded nucleic acid binding CSD and the C-terminal highly charged region. (C) RT–PCR analysis of RNAs produced upon transfection of HeLa cells with the reporter shown in (A) and increasing amounts (0, 1, 2, 3 or 4 µg) of either wild-type or mutant YB-1 expression plasmids. Duplicate lanes are shown for each concentration of the wild-type YB-1. PCR oligonucleotides were complementary to sequences in the flanking β-globin exons. Product RNAs resulting from inclusion of neither CD44 exon, either exon v4 or v5, or both exons v4 and v5 are indicated. Note that exons v4 and v5 have such similar sizes that inclusion bands resulting from either of the exons will be in the same region of the gel. Increasing amounts of the YB-1 expression plasmid resulted in a linear increase in the amount of YB-1 mRNA (data not shown). (D) Quantification of the effect of YB-1 on inclusion of CD44 exons v4 and v5. Results of four independent experiments are shown with 1 SD indicated.

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Transfection, Mutagenesis, Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Produced, Concentration Assay, Plasmid Preparation

Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 4. Antibodies to YB-1 peptides immunoprecipitate p50. Polyclonal antibodies raised against a C-terminal peptide of human YB-1 were used to immunoprecipitate proteins cross-linked to the ACE sel RNA. (A) Immunoprecipitation of cross-linking proteins in HeLa nuclear extract. Duplicate immunoprecipitations are shown. Lane 1, total cross-linking; lanes 2 and 3, supernatants for immunoprecipitations 1 and 2; lanes 4 and 5, pellets from immunoprecipitations 1 and 2. (B) Competition of UV cross-linking of nuclear proteins (lanes 1–3) or gel-purified and renatured recombinant human YB-1 (lanes 4–6). Cross-linking reactions were performed without competitor (lanes 1 and 4) or with 10 pmol of the ACE sel competitor (lanes 2 and 5) or the ACE sel Mut 1 competitor (lanes 3 and 6). The positions of HeLa YB-1 and recombinant YB-1 are indicated; the latter is slightly larger as a result of the presence of a His tag.

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Immunoprecipitation, Purification, Recombinant

Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 5. Human CD44 alternative splicing. (A) The exon–intron architecture of the human CD44 gene is drawn at the top. The 10 alternative cassette exons are depicted in red. The exon studied in this report is the fourth variable exon v4. The sequence of this exon is shown at the bottom of the figure. Sequences within the exon are indicated with a gray background. The red sequence indicates A/C-rich elements ACE 1, ACE 2 and ACE 3 within exon v4 that are potential binding sites for YB-1. (B) A/C-rich repeats within CD44 variable exons 4 and 5. The A/C-rich sequences from the two exons are aligned and a consensus repeat sequence (blue) is derived. At the top is the derived repeat consensus ACE from the exon selection experiments.

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Sequencing, Binding Assay, Derivative Assay, Selection

Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 6. CD44 exon v4 can be UV cross-linked to YB-1. (A) The substrate RNAs diagrammed below the gel were UV cross-linked in a standard splicing assay using HeLa nuclear extract. A diagram of the region of the human CD44 gene containing the fourth and fifth alternative exons is indicated below the gel. Intron 9 is the naturally occurring intron separating exons v4 and v5. ACE elements within exon v4 are indicated by white circles. The partial exon v4 RNAs shown in lanes 2 and 3 were generated by transcript termination at the indicated restriction sites. The ACE sel RNA used for lane 4 is longer than the RNA used for previous figures (see Materials and methods) resulting in the cross-linking of a protein slightly larger thanYB-1 in addition to YB-1. The position of YB-1 is indicated. (B) Immunoprecipitation of cross-linked proteins with an antibody specific for YB-1. A substrate RNA containing the first half of exon 4 and including the ACE 1 and ACE 2 elements (diagrammed in C) was subjected to UV cross-linking in a standard HeLa in vitro splicing assay. Cross-linked proteins were immunoprecipitated using the anti-YB-1 antibody and displayed by SDS–PAGE. Lane 1, total proteins; lane 2, supernatant; lane 3, precipitated protein (3-fold more of the reaction was loaded in lanes 2 and 3 than 1; lanes 2 and 3 came from the same reaction). (C) Competition of YB-1 cross-linking to CD44 exon v4 with an RNA containing ACE sel. Two substrate RNAs were employed: the exon 4 substrate described in (B) (lanes 1–4) and the ACE sel RNA itself (lanes 5–8). Competitor concentrations were 0, 0.3, 3.0 and 10 pmol. The position of cross-linked YB-1 is indicated.

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Splicing Assay, Generated, Immunoprecipitation, In Vitro, SDS Page

Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 10. Mutation of CD44 ACE sequences depresses UV cross-linking of YB-1. Top: the binding of YB-1 to wild-type and mutant CD44 exon 4 sequences was compared by UV cross-linking. The substrates are diagrammed above the gel. The position of YB-1 is indicated. Bottom: immunoprecipitation of YB-1 UV cross-linked to wild-type or mutant substrates. Cross-linked reactions were immunoprecipitated using the YB-1-specific antibody. Proteins from both the precipitate and the supernatant were displayed by SDS–PAGE.

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, SDS Page

Journal:

Article Title: The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4

doi: 10.1093/emboj/20.14.3821

Figure Lengend Snippet: Fig. 11. Mutation of CD44 ACE sequences depresses the ability to respond to YB-1 in vivo. (A) The mini-gene containing CD44 variable exons v4 and v5 was co-transfected with an expression vector coding for YB-1 as in Figure 9. The mini-gene contained a wild-type exon v4 (lanes 1–4) or an exon v4 containing the ACE 3 mutation (lanes 5–8). Increasing amounts of YB-1 vector were used (0, 1, 2 or 4 µg). The positions of RNA species resulting from the inclusion of no, one or two CD44 exons are indicated. (B) Quantification of the results in (A).

Article Snippet: To confirm the identification of p50 as YB-1, we prepared a rabbit polyclonal antibody to a C-terminal peptide of human YB-1, AENSSAPEAEQGGAE (Anaspec, CA).

Techniques: Mutagenesis, In Vivo, Transfection, Expressing, Plasmid Preparation